Mixtures of drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same

ABSTRACT

A non-polydispersed mixture of conjugates in which each conjugate in the mixture comprises a drug coupled to an oligomer that includes a polyalkylene glycol moiety is disclosed. The mixture may exhibit higher in vivo activity than a polydispersed mixture of similar conjugates. The mixture may be more effective at surviving an in vitro model of intestinal digestion than polydispersed mixtures of similar conjugates. The mixture may result in less inter-subject variability than polydispersed mixtures of similar conjugates.

FIELD OF THE INVENTION

[0001] The present invention relates to drug-oligomer conjugates.

BACKGROUND OF THE INVENTION

[0002] Pharmaceutically active molecules such as proteins andpolypeptides have been conjugated with polydispersed mixtures ofpolyethylene glycol or polydispersed mixtures of polyethylene glycolcontaining polymers to provide polydispersed mixtures of drug-oligomerconjugates. For example, U.S. Pat. No. 4,179,337 to Davis et al.proposes conjugating polypeptides such as insulin with variouspolyethylene glycols such as MPEG-1900 and MPEG-5000 supplied by UnionCarbide.

[0003] U.S. Pat. No. 5,567,422 to Greenwald proposes the conjugation ofbiologically active nucleophiles with polyethylene glycols such asm-PEG-OH (Union Carbide), which has a number average molecular weight of5,000 Daltons.

[0004] U.S. Pat. No. 5,405,877 to Greenwald et al. proposes reactingbovine hemoglobin with thiazolidine thione activated PEG, which wasprepared using m-PEG carboxylic acid having a number average molecularweight of 5,000 Daltons.

[0005] U.S. Pat. No. 5,359,030 to Ekwuribe proposes conjugatingpolypeptides such as insulin with polyethylene glycol modifiedglycolipid polymers and polyethylene glycol modified fatty acidpolymers. In this patent, the number average molecular weight of polymerresulting from each combination is preferred to be in the range of fromabout 500 to about 10,000 Daltons.

[0006] PEG is typically produced by base-catalyzed ring-openingpolymerization of ethylene oxide. The reaction is initiated by addingethylene oxide to ethylene glycol, with potassium hydroxide as catalyst.This process results in a polydispersed mixture of polyethylene glycolpolymers having a number average molecular weight within a given rangeof molecular weights. For example, PEG products offered by Sigma-Aldrichof Milwaukee, Wis. are provided in polydispersed mixtures such as PEG400 (M_(n) 380-420); PEG 1,000 (M_(n) 950-1,050); PEG 1,500 (M_(n)1,400-1,600); and PEG 2,000 (M_(n) 1,900-2,200).

[0007] It is desirable to provide non-polydispersed mixtures ofdrug-oligomer conjugates that comprises polyalkylene glycol.

SUMMARY OF THE INVENTION

[0008] A mixture of drug-oligomer conjugates comprising polyalkyleneglycol according to embodiments of the present invention may exhibithigher in vivo activity than a polydispersed mixture of similarconjugates, where the polydispersed mixture has the same number averagemolecular weight as the mixture according to the present invention. Thisheightened activity may result in lower dosage requirements. Moreover, amixture of drug-oligomer conjugates comprising polyalkylene glycolaccording to embodiments of the present invention may be more effectiveat surviving an in vitro model of intestinal digestion thanpolydispersed mixtures of similar conjugates. Furthermore, a mixture ofdrug-oligomer conjugates comprising polyalkylene glycol according toembodiments of the present invention may result in less inter-subjectvariability than polydispersed mixtures of similar conjugates.

[0009] According to embodiments of the present invention, asubstantially monodispersed mixture of conjugates each comprising a drugcoupled to an oligomer that comprises a polyalkylene glycol moiety isprovided. The mixture preferably is a monodispersed mixture and, morepreferably, is a purely monodispersed mixture. The polyalkylene glycolmoiety preferably has at least 2, 3 or 4 polyalkylene glycol subunits.Most preferably, the polyalkylene glycol moiety preferably has at least7 polyalkylene glycol subunits. The polyalkylene glycol moiety ispreferably a polyethylene glycol moiety or polypropylene glycol moiety.The oligomer preferably further comprises a lipophilic moiety. Theconjugate is preferably amphiphilically balanced such that the conjugateis aqueously soluble and able to penetrate biological membranes. Theoligomer may comprise a first polyalkylene glycol moiety covalentlycoupled to the drug by a non-hydrolyzable bond and a second polyalkyleneglycol moiety covalently coupled to the first polyalkylene glycol moietyby a hydrolyzable bond.

[0010] According to other embodiments of the present invention, asubstantially monodispersed mixture of conjugates is provided where eachconjugate comprises a drug coupled to an oligomer including apolyalkylene glycol moiety, and the mixture has an in vivo activity thatis greater than the in vivo activity of a polydispersed mixture ofdrug-oligomer conjugates having the same number average molecular weightas the substantially monodispersed mixture.

[0011] According to still other embodiments of the present invention, asubstantially monodispersed mixture of conjugates is provided where eachconjugate comprises a drug coupled to an oligomer including apolyalkylene glycol moiety, and the mixture has an in vitro activitythat is greater than the in vitro activity of a polydispersed mixture ofdrug-oligomer conjugates having the same number average molecular weightas the substantially monodispersed mixture.

[0012] According to other embodiments of the present invention, asubstantially monodispersed mixture of conjugates is provided where eachconjugate comprises a drug coupled to an oligomer including apolyalkylene glycol moiety, and the mixture has an increased resistanceto degradation by chymotrypsin when compared to the resistance todegradation by chymotrypsin of a polydispersed mixture of drug-oligomerconjugates having the same number average molecular weight as thesubstantially monodispersed mixture.

[0013] According to yet other embodiments of the present invention, asubstantially monodispersed mixture of conjugates is provided where eachconjugate comprises a drug coupled to an oligomer including apolyalkylene glycol moiety, and the mixture has an inter-subjectvariability that is less than the inter-subject variability of apolydispersed mixture of drug-oligomer conjugates having the same numberaverage molecular weight as the substantially monodispersed mixture.

[0014] According to still other embodiments of the present invention, amixture of conjugates is provided where each conjugate includes a drugcoupled to an oligomer that comprises a polyalkylene glycol moiety, andthe mixture has a molecular weight distribution with a standarddeviation of less than about 22 Daltons.

[0015] According to yet other embodiments of the present invention, amixture of conjugates is provided where each conjugate includes a drugcoupled to an oligomer that comprises a polyalkylene glycol moiety, andthe mixture has a dispersity coefficient (DC) greater than 10,000 where${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}\quad N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}}} \right)^{2}}$

[0016] wherein:

[0017] n is the number of different molecules in the sample;

[0018] N_(i) is the number of i^(th) molecules in the sample; and

[0019] M_(i) is the mass of the i^(th) molecule.

[0020] According to other embodiments of the present invention, amixture of conjugates is provided in which each conjugate includes adrug coupled to an oligomer and has the same number of polyalkyleneglycol subunits.

[0021] According to still other embodiments of the present invention, amixture of conjugates is provided in which each conjugate has the samemolecular weight and has the formula:

[0022] wherein:

[0023] B is a bonding moiety;

[0024] L is a linker moiety;

[0025] G, G′ and G″ are individually selected spacer moieties;

[0026] R is a lipophilic moiety and R′ is a polyalkylene glycol moiety,or R′ is the lipophilic moiety and R is the polyalkylene glycol moiety;

[0027] T is a terminating moiety;

[0028] h, i, j, k, m and n are individually 0 or 1, with the provisothat when R is the polyalkylene glycol moiety; m is 1, and when R′ isthe polyalkylene glycol moiety, n is 1; and

[0029] p is an integer from 1 to the number of nucleophilic residues onthe drug.

[0030] Pharmaceutical compositions comprising conjugate mixtures of thepresent invention as well as methods of treating a disease state in asubject in need of such treatment by administering an effective amountof such pharmaceutical compositions are also provided. Additionally,methods of synthesizing such conjugate mixtures are provided.

[0031] Drug-oligomer conjugate mixtures according to embodiments of thepresent invention may provide increased in vivo activity and/or loweredinter-subject variability and/or decreased degradation by chymotrypsinwhen compared to conventional polydispersed drug-oligomer conjugatemixtures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 illustrates a generic scheme for synthesizing a mixture ofactivated polymers comprising a polyethylene glycol moiety and a fattyacid moiety according to embodiments of the present invention;

[0033]FIG. 2 illustrates a scheme for synthesizing a mixture of mPEGaccording to embodiments of the present invention;

[0034]FIG. 3 illustrates a scheme for synthesizing a mixture ofactivated mPEG7-hexyl oligomers according to embodiments of the presentinvention;

[0035]FIG. 4 illustrates a scheme for synthesizing a mixture ofactivated mPEG7-octyl oligomers according to embodiments of the presentinvention;

[0036]FIG. 5 illustrates a scheme for synthesizing a mixture ofactivated mPEG-decyl oligomers according to embodiments of the presentinvention;

[0037]FIG. 6 illustrates a scheme for synthesizing a mixture ofactivated stearate-PEG6 oligomers according to embodiments of thepresent invention;

[0038]FIG. 7 illustrates a scheme for synthesizing a mixture ofactivated stearate-PEG8 oligomers according to embodiments of thepresent invention;

[0039]FIG. 8 illustrates a scheme for synthesizing a mixture ofactivated PEG3 oligomers according to embodiments of the presentinvention;

[0040]FIG. 9 illustrates a scheme for synthesizing a mixture ofactivated palmitate-PEG3 oligomers according to embodiments of thepresent invention;

[0041]FIG. 10 illustrates a scheme for synthesizing a mixture ofactivated PEG6 oligomers and conjugating human growth hormone with theactivated PEG6 oligomers according to embodiments of the presentinvention;

[0042]FIG. 11 illustrates a scheme for synthesizing various propyleneglycol monomers according to embodiments of the present invention;

[0043]FIG. 12 illustrates a scheme for synthesizing various propyleneglycol polymers according to embodiments of the present invention;

[0044]FIG. 13 illustrates a scheme for synthesizing various propyleneglycol polymers according to embodiments of the present invention;

[0045]FIG. 14 is an HPLC trace (HPLC gradient: 50% to 90% acetonitrilein 30 minutes) of the conjugation reaction illustrated in FIG. 10 using2 equivalents of activated MPEG6 oligomers and 5 equivalents ofactivated MPEG6 oligomers;

[0046]FIG. 15 is an HPLC trace (HPLC gradient: 0% to 95% acetonitrile in20 minutes) of the conjugation reaction illustrated in FIG. 10 using 30equivalents of activated MPEG6 oligomers;

[0047]FIG. 16 is a MALDI spectra of the conjugation reaction illustratedin FIG. 10 using 2 equivalents of activated MPEG6 oligomers;

[0048]FIG. 17 is an HPLC trace (HPLC gradient: 50% to 70% acetonitrilein 30 minutes) illustrating a partial purification of the product of theconjugation reaction of FIG. 10 using 5 equivalents of activated MPEG6oligomers;

[0049]FIG. 18 is a MALDI spectra of fraction B from the partialpurification illustrated in FIG. 17;

[0050]FIG. 19 is a MALDI spectra of fraction C from the partialpurification illustrated in FIG. 17;

[0051]FIG. 20 is a MALDI spectra of fractions D and E from the partialpurification illustrated in FIG. 17;

[0052]FIG. 21 is an electrospray spectra of fraction E from the partialpurification illustrated in FIG. 17;

[0053]FIG. 22 is an electrospray spectra of the reaction mixture fromthe conjugation reaction illustrated in FIG. 10 using 30 equivalents ofactivated MPEG6 oligomers;

[0054]FIG. 23 is an HPLC trace of a conjugation reaction of human growthhormone with the activated oligomer of FIG. 9;

[0055]FIG. 24 is an HPLC trace of a conjugation reaction using oneequivalent of human growth hormone and two equivalents of the activatedoligomer of FIG. 9 according to the present invention compared with anHPLC trace of human growth hormone, which does not form part of thepresent invention;

[0056]FIG. 25 is an HPLC trace of a conjugation reaction using oneequivalent of human growth hormone and five equivalents of the activatedoligomer of FIG. 8 according to the present invention compared with anHPLC trace of human growth hormone, which does not form part of thepresent invention;

[0057]FIG. 26 is a MALDI spectra of the fraction corresponding to theleft half of the peak in the conjugation HPLC trace of FIG. 25;

[0058]FIG. 27 is a MALDI spectra of the fraction corresponding to theright half of the peak in the conjugation HPLC trace of FIG. 25;

[0059]FIG. 28 is an HPLC trace of a conjugation reaction using oneequivalent of human growth hormone and 9 equivalents of the activatedoligomer of FIG. 8 according to the present invention compared with anHPLC trace of human growth hormone, which does not form part of thepresent invention;

[0060]FIG. 29 illustrates a comparison of results obtained with aCytosensor® Microphysiometer, which provides an indication of theactivity of a compound, for mixtures of insulin-oligomer conjugatesaccording to embodiments of the present invention compared withpolydispersed conjugate mixtures and insulin, which are provided forcomparison purposes only and do not form part of the invention;

[0061]FIG. 30 illustrates a comparison of chymotrypsin degradation ofinsulin-oligomer conjugates according to embodiments of the presentinvention with a conventional polydispersed mixture of insulin-oligomerconjugates, which is provided for comparison purposes only and does notform part of the invention;

[0062]FIG. 31 illustrates the effect of a mixture ofmPEG7-hexyl-insulin, monoconjugate, according to embodiments of thepresent invention on plasma glucose in fasted beagles;

[0063]FIG. 32 illustrates, for comparison purposes, the effect of apolydispersed mixture of mPEG7_(avg)-hexyl-insulin, monoconjugate, whichis not part of the present invention, on plasma glucose in fastedbeagles;

[0064]FIG. 33 illustrates the inter-subject variability of a mixture ofmPEG4-hexyl-insulin monoconjugates according to embodiments of thepresent invention administered to fasted beagles;

[0065]FIG. 34 illustrates the inter-subject variability of a mixture ofmPEG7-hexyl-insulin monoconjugates according to embodiments of thepresent invention administered to fasted beagles;

[0066]FIG. 35 illustrates the inter-subject variability for a mixture ofmPEG10-hexyl-insulin monoconjugates according to embodiments of thepresent invention administered to fasted beagles;

[0067]FIG. 36 illustrates, for comparison purposes, the inter-subjectvariability of a polydispersed mixture of mPEG7_(avg)-hexyl-insulinmonoconjugates, which is not part of the present invention, administeredto fasted beagles;

[0068]FIG. 37 illustrates a comparison of the average AUCs for variousmonodispersed mixtures of calcitonin-oligomer conjugates according toembodiments of the present invention with non-conjugated calcitonin,which is provided for comparison purposes only and does not form part ofthe invention;

[0069]FIG. 38 illustrates dose-response curves where the response ismeasured as a percentage of the maximum possible response for a mixtureof mPEG7-octyl-calcitonin diconjugates according to embodiments of thepresent invention compared with calcitonin, which is provided forcomparison purposes and is not a part of the present invention;

[0070]FIG. 39 illustrates a dose-response curve after oraladministration of a mixture of mPEG7-octyl-calcitonin diconjugatesaccording to embodiments of the present invention;

[0071]FIG. 40 illustrates a dose-response curve after subcutaneousadministration of a mixture of mPEG7-octyl-calcitonin diconjugatesaccording to embodiments of the present invention;

[0072]FIG. 41 illustrates a dose-response curve after subcutaneousadministration of salmon calcitonin, which is provided for comparisonpurposes and is not part of the present invention;

[0073]FIG. 42 illustrates a bar graph denoting the activity asdetermined by luciferase assay of mixtures of growth hormone conjugatesaccording to embodiments of the present invention compared with theactivity of human growth hormone standards, which are provided forcomparison purposes only and do not form part of the present invention;and

[0074]FIG. 43 illustrates a bar graph denoting the activity asdetermined by luciferase assay of mixtures of growth hormone conjugatesaccording to embodiments of the present invention compared with theactivity of human growth hormone standards, which are provided forcomparison purposes only and do not form part of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0075] The invention will now be described with respect to preferredembodiments described herein. It should be appreciated however thatthese embodiments are for the purpose of illustrating the invention, andare not to be construed as limiting the scope of the invention asdefined by the claims.

[0076] As used herein, the term “non-polydispersed” is used to describea mixture of compounds having a dispersity that is in contrast to thepolydispersed mixtures described in U.S. Pat. No. 4,179,337 to Davis etal.; U.S. Pat. No. 5,567,422 to Greenwald; U.S. Pat. No. 5,405,877 toGreenwald et al.; and U.S. Pat. No. 5,359,030 to Ekwuribe.

[0077] As used herein, the term “substantially monodispersed” is used todescribe a mixture of compounds wherein at least about 95 percent of thecompounds in the mixture have the same molecular weight.

[0078] As used herein, the term “monodispersed” is used to describe amixture of compounds wherein about 100 percent of the compounds in themixture have the same molecular weight.

[0079] As used herein, the term “substantially purely monodispersed” isused to describe a mixture of compounds wherein at least about 95percent of the compounds in the mixture have the same molecular weightand same molecular structure. Thus, a substantially purely monodispersedmixture is a substantially monodispersed mixture, but a substantiallymonodispersed mixture is not necessarily a substantially purelymonodispersed mixture.

[0080] As used herein, the term “purely monodispersed” is used todescribe a mixture of compounds wherein about 100 percent of thecompounds in the mixture have the same molecular weight and have thesame molecular structure. Thus, a purely monodispersed mixture is amonodispersed mixture, but a monodispersed mixture is not necessarily apurely monodispersed mixture.

[0081] As used herein, the term “weight average molecular weight” isdefined as the sum of the products of the weight fraction for a givenmolecule in the mixture times the mass of the molecule for each moleculein the mixture. The “weight average molecular weight” is represented bythe symbol M_(w).

[0082] As used herein, the term “number average molecular weight” isdefined as the total weight of a mixture divided by the number ofmolecules in the mixture and is represented by the symbol M_(n).

[0083] As used herein, the term “dispersity coefficient” (DC) is definedby the formula:${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}\quad N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}}} \right)^{2}}$

[0084] wherein:

[0085] n is the number of different molecules in the sample;

[0086] N_(i) is the number of i^(th) molecules in the sample; and

[0087] M_(i) is the mass of the i^(th) molecule.

[0088] As used herein, the term “intra-subject variability” means thevariability in activity occurring within the same subject when thesubject is administered the same dose of a drug or pharmaceuticalcomposition at different times.

[0089] As used herein, the term “inter-subject variability” means thevariability in activity between two or more subjects when each subjectis administered the same dose of a given drug or pharmaceuticalformulation.

[0090] As used herein, the term “polyalkylene glycol” refers to straightor branched polyalkylene glycol polymers such as polyethylene glycol,polypropylene glycol, and polybutylene glycol, and includes themonoalkylether of the polyalkylene glycol. The term “polyalkylene glycolsubunit” refers to a single polyalkylene glycol unit. For example, apolyethylene glycol subunit would be —O—CH₂—CH₂—O—.

[0091] As used herein, the term “lipophilic” means the ability todissolve in lipids and/or the ability to penetrate, interact with and/ortraverse biological membranes, and the term, “lipophilic moiety” or“lipophile” means a moiety which is lipophilic and/or which, whenattached to another chemical entity, increases the lipophilicity of suchchemical entity. Examples of lipophilic moieties include, but are notlimited to, alkyls, fatty acids, esters of fatty acids, cholesteryl,adamantyl and the like.

[0092] As used herein, the term “lower alkyl” refers to substituted orunsubstituted alkyl moieties having from 1 to 5 carbon atoms.

[0093] As used herein, the term “higher alkyl” refers to substituted orunsubstituted alkyl moieties having 6 or more carbon atoms.

[0094] As used herein, the term “drug” refers to any therapeuticcompound that is conjugatable in the manner of the present invention.Representative non-limiting classes of therapeutic compounds useful inthe present invention include those falling into the followingtherapeutic categories: ACE-inhibitors; anti-anginal drugs;anti-arrhythmias; anti-asthmatics; anti-cholesterolemics;anti-convulsants; anti-depressants; anti-diarrhea preparations;anti-histamines; anti-hypertensive drugs; anti-infectives;anti-inflammatory agents; anti-lipid agents; anti-manics;anti-nauseants; anti-stroke agents; anti-thyroid preparations;anti-tumor drugs; anti-tussives; anti-uricemic drugs; anti-viral agents;acne drugs; alkaloids; amino acid preparations; anabolic drugs;analgesics; anesthetics; angiogenesis inhibitors; antacids;anti-arthritics; antibiotics; anticoagulants; antiemetics; antiobesitydrugs; antiparasitics; antipsychotics; antipyretics; antispasmodics;antithrombotic drugs; anxiolytic agents; appetite stimulants; appetitesuppressants; beta blocking agents; bronchodilators; cardiovascularagents; cerebral dilators; chelating agents; cholecystokininantagonists; chemotherapeutic agents; cognition activators;contraceptives; coronary dilators; cough suppressants; decongestants;deodorants; dermatological agents; diabetes agents; diuretics;emollients; enzymes; erythropoietic drugs; expectorants; fertilityagents; fungicides; gastrointestinal agents; growth regulators; hormonereplacement agents; hyperglycemic agents; hypnotics; hypoglycemicagents; laxatives; migraine treatments; mineral supplements; mucolytics;narcotics; neuroleptics; neuromuscular drugs; NSAIDS; nutritionaladditives; peripheral vasodilators; polypeptides; prostaglandins;psychotropics; renin inhibitors; respiratory stimulants; steroids;stimulants; sympatholytics; thyroid preparations; tranquilizers; uterinerelaxants; vaginal preparations; vasoconstrictors; vasodilators; vertigoagents; vitamins; and wound healing agents.

[0095] The drug is preferably a polypeptide. Non-limiting examples ofpolypeptides that may be useful in the present invention include thefollowing:

[0096] Adrenocorticotropic hormone (ACTH) peptides including, but notlimited to, ACTH, human; ACTH 1-10; ACTH 1-13, human; ACTH 1-16, human;ACTH 1-17; ACTH 1-24, human; ACTH 4-10; ACTH 4-11; ACTH 6-24; ACTH 7-38,human; ACTH 18-39, human; ACTH, rat; ACTH 12-39, rat; beta-cell tropin(ACTH 22-39); biotinyl-ACTH 1-24, human; biotinyl-ACTH 7-38, human;corticostatin, human; corticostatin, rabbit; [Met(02)⁴, DLys⁸, Phe⁹]ACTH 4-9, human; [Met(0)⁴,DLys⁸, Phe⁹] ACTH 4-9, human; N-acetyl, ACTH1-17, human; and ebiratide.

[0097] Adrenomedullin peptides including, but not limited to,adrenomedullin, adrenomedullin 1-52, human; adrenomedullin 1-12, human;adrenomedullin 13-52, human; adrenomedullin 22-52, human;pro-adrenomedullin 45-92, human; pro-adrenomedullin 153-185, human;adrenomedullin 1-52, porcine; pro-adrenomedullin (N-20), porcine;adrenomedullin 1-50, rat; adrenomedullin 11-50, rat; and proAM-N20(proadrenomedullin N-terminal 20 peptide), rat.

[0098] Allatostatin peptides including, but not limited to, allatostatinI; allatostatin II; allatostatin III; and allatostatin IV.

[0099] Amylin peptides including, but not limited to, acetyl-amylin8-37, human; acetylated amylin 8-37, rat; AC187 amylin antagonist; AC253amylin antagonist; AC625 amylin antagonist; amylin 8-37, human; amylin(IAPP), cat; amylin (insulinoma or islet amyloid polypeptide(IAPP));amylin amide, human; amylin 1-13 (diabetes-associated peptide 1-13),human; amylin 20-29 (IAPP 20-29), human; AC625 amylin antagonist; amylin8-37, human; amylin (IAPP), cat; amylin, rat; amylin 8-37, rat;biotinyl-amylin, rat; and biotinyl-amylin amide, human.

[0100] Amyloid beta-protein fragment peptides including, but not limitedto, Alzheimer's disease beta-protein 12-28 (SP17); amyloid beta-protein25-35; amyloid beta/A4-protein precursor 328-332; amyloid beta/A4protein precursor (APP) 319-335; amyloid beta-protein 1-43; amyloidbeta-protein 1-42; amyloid beta-protein 1-40; amyloid beta-protein10-20; amyloid beta-protein 22-35; Alzheimer's disease beta-protein(SP28); beta-amyloid peptide 1-42, rat; beta-amyloid peptide 1-40, rat;beta-amyloid 1 - 11; beta-amyloid 31-35; beta-amyloid 32-35;beta-amyloid 35-25; beta-amyloid/A4 protein precursor 96-110;beta-amyloid precursor protein 657-676; beta-amyloid 1-38;[Gln¹¹]-Alzheimer's disease beta-protein; [Gln¹¹]-beta-amyloid 1-40;[Gln²²]-beta-amyloid 6-40; non-A beta component of Alzheimer's diseaseamyloid (NAC); P3, (A beta 17-40) Alzheimer's disease amyloid β-peptide;and SAP (serum amyloid P component) 194-204.

[0101] Angiotensin peptides including, but not limited to, A-779;Ala-Pro-Gly-angiotensin II; [Ile³,Val⁵]-angiotensin II; angiotensin IIIantipeptide; angiogenin fragment 108-122; angiogenin fragment 108-123;angiotensin I converting enzyme inhibitor; angiotensin I, human;angiotensin I converting enzyme substrate; angiotensin 11-7, human;angiopeptin; angiotensin II, human; angiotensin II antipeptide;angiotensin II 1-4, human; angiotensin II 3-8, human; angiotensin II4-8, human; angiotensin II 5-8, human; angiotensin III([Des-Asp¹]-angiotensin II), human; angiotensin III inhibitor([Ile⁷]-angiotensin III); angiotensin-converting enzyme inhibitor(Neothunnus macropterus); [Asn¹, Val⁵]-angiotensin I, goosefish; [Asn¹,Val⁵, Asn⁹]-angiotensin I, salmon; [Asn¹, Val⁵, Gly⁹]-angiotensin I,eel; [Asn¹, Val⁵]-angiotensin I 1-7, eel, goosefish, salmon; [Asn¹,Val⁵]-angiotensin II; biotinyl-angiotensin I, human;biotinyl-angiotensin II, human; biotinyl-Ala-Ala-Ala-angiotensin II;[Des-Asp¹]-angiotensin I, human; [p-aminophenylalanine⁶]-angiotensin II;renin substrate (angiotensinogen 1-13), human; preangiotensinogen 1-14(renin substrate tetradecapeptide), human; renin substratetetradecapeptide (angiotensinogen 1-14), porcine; [Sar¹]-angiotensin II,[Sar¹]-angiotensin II 1-7 amide; [Sar¹, Ala⁸]-angiotensin II; [Sar¹,Ile⁸]-angiotensin II; [Sar¹, Thr⁸]-angiotensin U; [Sar¹,Tyr(Me)⁴]-angiotensin II (Sarmesin); [Sar¹, Val⁵, Ala⁸]-angiotensin II;[Sar¹, Ile⁷]-angiotensin III; synthetic tetradecapeptide renin substrate(No. 2); [Val⁴]-angiotensin III; [Val⁵]-angiotensin II;[Val⁵]-angiotensin I, human; [Val⁵]-angiotensin I; [Val⁵,Asn⁹]-angiotensin I, bullfrog; and [Val⁵, Ser⁹]-angiotensin I, fowl.

[0102] Antibiotic peptides including, but not limited to, Ac-SQNY;bactenecin, bovine; CAP 37 (20-44);carbormethoxycarbonyl-DPro-DPhe-OBzl; CD36 peptide P 139-155; CD36peptide P 93-110; cecropin A-melittin hybrid peptide [CA(1-7)M(2-9)NH2];cecropin B, free acid; CYS(Bzl)84 CD fragment 81-92; defensin (human)HNP-2; dermaseptin; immunostimulating peptide, human; lactoferricin,bovine (BLFC); and magainin spacer.

[0103] Antigenic polypeptides, which can elicit an enhanced immuneresponse, enhance an immune response and or cause an immunizinglyeffective response to diseases and/or disease causing agents including,but not limited to, adenoviruses; anthrax; Bordetella pertussus;botulism; bovine rhinotracheitis; Branhamella catarrhalis; caninehepatitis; canine distemper; Chlamydiae; cholera; coccidiomycosis;cowpox; cytomegalovirus; Dengue fever; dengue toxoplasmosis; diphtheria;encephalitis; enterotoxigenic E. coli; Epstein Barr virus; equineencephalitis; equine infectious anemia; equine influenza; equinepneumonia; equine rhinovirus; Escherichia coli; feline leukemia;flavivirus; globulin; haemophilus influenza type b; Haemophilusinfluenzae; Haemophilus pertussis; Helicobacter pylon; hemophilus;hepatitis; hepatitis A; hepatitis B; Hepatitis C; herpes viruses; HIV;HIV-1 viruses; HIV-2 viruses; HTLV; influenza; Japanese encephalitis;Klebsiellae species; Legionella pneumophila; leishmania; leprosy; lymedisease; malaria immunogen; measles; meningitis; meningococcal;Meningococcal polysaccharide group A; Meningococcal polysaccharide groupC; mumps; mumps virus; mycobacteria; Mycobacterium tuberculosis;Neisseria; Neisseria gonorrhea; Neisseria meningitidis; ovine bluetongue; ovine encephalitis; papilloma; parainfluenza; paramyxoviruses;Pertussis; plague; pneumococcus; Pneumocystis carinii; pneumonia;poliovirus; proteus species; Pseudomonas aeruginosa; rabies; respiratorysyncytial virus; rotavirus; rubella; salmonellae; schistosomiasis;shigellae; simian immunodeficiency virus; smallpox; Staphylococcusaureus; Staphylococcus species; Streptococcus pneumoniae; Streptococcuspyogenes; Streptococcus species; swine influenza; tetanus; Treponemapallidum; typhoid; vaccinia; varicella-zoster virus; and vibriocholerae.

[0104] Anti-microbial peptides including, but not limited to, buforin I;buforin II; cecropin A; cecropin B; cecropin P1, porcine; gaegurin 2(Rana rugosa); gaegurin 5 (Rana rugosa); indolicidin; protegrin-(PG)-I;magainin 1; and magainin 2; and T-22 [Tyr^(5,12), Lys⁷]-poly-phemusin IIpeptide.

[0105] Apoptosis related peptides including, but not limited to,Alzheimer's disease beta-protein (SP28); calpain inhibitor peptide;capsase-1 inhibitor V; capsase-3, substrate IV; caspase-1 inhibitor I,cell-permeable; caspase-1 inhibitor VI; caspase-3 substrate III,fluorogenic; caspase-1 substrate V, fluorogenic; caspase-3 inhibitor I,cell-permeable; caspase-6 ICE inhibitor III; [Des-Ac, biotin]-ICEinhibitor III; IL-1 B converting enzyme (ICE) inhibitor II; IL-I Bconverting enzyme (ICE) substrate IV; MDL 28170; and MG-132.

[0106] Atrial natriuretic peptides including, but not limited to,alpha-ANP (alpha-chANP), chicken; anantin; ANP 1-11, rat; ANP 8-30,frog; ANP 11-30, frog; ANP-21 (fANP-21), frog; ANP-24 (fANP-24), frog;ANP-30, frog; ANP fragment 5-28, human, canine; ANP-7-23, human; ANPfragment 7-28, human, canine; alpha-atrial natriuretic polypeptide 1-28,human, canine; A71915, rat; atrial natriuretic factor 8-33, rat; atrialnatriuretic polypeptide 3-28, human; atrial natriuretic polypeptide4-28, human, canine; atrial natriuretic polypeptide 5-27; human; atrialnatriuretic aeptide (ANP), eel; atriopeptin I, rat, rabbit, mouse;atriopeptin II, rat, rabbit, mouse; atriopeptin III, rat, rabbit, mouse;atrial natriuretic factor (rANF), rat, auriculin A (rat ANF 126-149);auriculin B (rat ANF 126-150); beta-ANP (1-28, dimer, antiparallel);beta-rANF 17-48; biotinyl-alpha-ANP 1-28, human, canine; biotinyl-atrialnatriuretic factor (biotinyl-rANF), rat; cardiodilatin 1-16, human;C-ANF 4-23, rat; Des-[Cys¹⁰⁵, Cys¹²¹]-atrial natriuretic factor 104-126,rat; [Met(O)¹²] ANP 1-28, human; [Mpr⁷,DAla⁹]ANP 7-28, amide, rat;prepro-ANF 104-116, human; prepro-ANF 26-55 (proANF 1-30), human;prepro-ANF 56-92 (proANF 31-67), human; prepro-ANF 104-123, human;[Tyr⁰]-atriopeptin I, rat, rabbit, mouse; [Tyr⁰]-atriopeptin II, rat,rabbit, mouse; [Tyr⁰]-prepro ANF 104-123, human; urodilatin (CDD/ANP95-126); ventricular natriuretic peptide (VNP), eel; and ventricularnatriuretic peptide (VNP), rainbow trout.

[0107] Bag cell peptides including, but not limited to, alpha bag cellpeptide; alpha-bag cell peptide 1-9; alpha-bag cell peptide 1-8;alpha-bag cell peptide 1-7; beta-bag cell factor; and gamma-bag cellfactor.

[0108] Bombesin peptides including, but not limited to, alpha-s1 casein101-123 (bovine milk); biotinyl-bombesin; bombesin 8-14; bombesin;[Leul³-psi (CH2NH)Leu¹⁴]-bombesin; [D-Phe⁶, Des-Met¹⁴]-bombesin 6-14ethylamide; [DPhe¹²] bombesin; [DPhe¹²,Leu¹⁴]-bombesin; [Tyr⁴]-bombesin;and [Tyr4,DPhe¹²]-bombesin.

[0109] Bone GLA peptides (BGP) including, but not limited to, bone GLAprotein; bone GLA protein 45-49; [Glu¹⁷, Gla^(21,24)]-osteocalcin 1-49,human; myclopeptide -2 (MP-2); osteocalcin 1-49 human; osteocalcin37-49, human; and [Tyr³⁸, Phe^(42,46)] bone GLA protein 38-49, human.

[0110] Bradykinin peptides including, but not limited to, [Ala^(2,6),des-Pro³]-bradykinin; bradykinin; bradykinin (Bowfin. Gar); bradykininpotentiating peptide; bradykinin 1-3; bradykinin 1-5; bradykinin 1-6;bradykinin 1-7; bradykinin 2-7; bradykinin 2-9; [DPhe⁷] bradykinin;[Des-Arg⁹]-bradykinin; [Des-Arg¹⁰]-Lys-bradykinin([Des-Arg¹⁰]-kallidin); [D-N-Me-Phe⁷]-bradykinin; [Des-Arg⁹,Leu⁸]-bradykinin; Lys-bradykinin (kallidin);Lys-[Des-Arg⁹,Leu⁸]-bradykinin ([Des-Arg¹⁰,Leu⁹]-kallidin); [Lys⁰-Hyp³]-bradykinin; ovokinin; [Lys⁰, Ala³]-bradykinin; Met-Lys-bradykinin;peptide K12 bradykinin potentiating peptide;[(pCl)Phe^(5,8)]-bradykinin; T-kinin (Ile-Ser-bradykinin); [Thi^(5,8),D-Phe⁷]-bradykinin; [Tyr⁰]-bradykinin; [Tyr⁵]-bradykinin;[Tyr⁸]-bradykinin; and kallikrein.

[0111] Brain natriuretic peptides (BNP) including, but not limited to,BNP 32, canine; BNP-like Peptide, eel; BNP-32, human; BNP-45, mouse;BNP-26, porcine; BNP-32, porcine; biotinyl-BNP-32, porcine; BNP-32, rat;biotinyl-BNP-32, rat; BNP-45 (BNP 51-95, 5K cardiac natriureticpeptide), rat; and [Tyr⁰]-BNP 1-32, human.

[0112] C-peptides including, but not limited to, C-peptide; and[Tyr⁰]-C-peptide, human.

[0113] C-type natriuretic peptides (CNP) including, but not limited to,C-type natriuretic peptide, chicken; C-type natriuretic peptide-22(CNP-22), porcine, rat, human; C-type natriuretic peptide-53 (CNP-53),human; C-type natriuretic peptide-53 (CNP-53), porcine, rat; C-typenatriuretic peptide-53 (porcine, rat) 1-29 (CNP-53 1-29); prepro-CNP1-27, rat; prepro-CNP 30-50, porcine, rat; vasonatrin peptide (VNP); and[Tyr⁰]-C-type natriuretic peptide-22 ([Tyr⁰]-CNP-22).

[0114] Calcitonin peptides including, but not limited to,biotinyl-calcitonin, human; biotinyl-calcitonin, rat;biotinyl-calcitonin, salmon; calcitonin, chicken; calcitonin, eel;calcitonin, human; calcitonin, porcine; calcitonin, rat; calcitonin,salmon; calcitonin 1-7, human; calcitonin 8-32, salmon; katacalcin(PDN-21) (C-procalcitonin); and N-proCT (amino-terminal procalcitonincleavage peptide), human.

[0115] Calcitonin gene related peptides (CGRP) including, but notlimited to, acetyl-alpha-CGRP 19-37, human; alpha-CGRP 19-37, human;alpha-CGRP 23-37, human; biotinyl-CGRP, human; biotinyl-CGRP II, human;biotinyl-CGRP, rat; beta-CGRP, rat; biotinyl-beta-CGRP, rat; CGRP, rat;CGRP, human; calcitonin C-terminal adjacent peptide; CGRP 1-19, human;CGRP 20-37, human; CGRP 8-37, human; CGRP II, human; CGRP, rat; CGRP8-37, rat; CGRP 29-37, rat; CGRP 30-37, rat; CGRP 31-37, rat; CGRP32-37, rat; CGRP 33-37, rat; CGRP 31-37, rat; ([Cys(Acm)^(2,7)]-CGRP;elcatonin; [Tyr⁰]-CGRP, human; [Tyr⁰]-CGRP II, human; [Tyr⁰]-CGRP 28-37,rat; [Tyr⁰]-CGRP, rat; and [Tyr²²]-CGRP 22-37, rat.

[0116] CART peptides including, but not limited to, CART, human; CART55-102, human; CART, rat; and CART 55-102, rat.

[0117] Casomorphin peptides including, but not limited to,beta-casomorphin, human; beta-casomorphin 1-3; beta-casomorphin 1-3,amide; beta-casomorphin, bovine; beta-casomorphin 1-4, bovine;beta-casomorphin 1-5, bovine; beta-casomorphin 1-5, amide, bovine;beta-casomorphin 1-6, bovine; [DAla²]-beta-casomorphin 1-3, amide,bovine; [DAla²,Hyp⁴,Tyr⁵]-beta-casomorphin 1-5 amide;[DAla²,DPro⁴,Tyr⁵]-beta-casomorphin 1-5, amide;[DAla²,Tyr⁵]-beta-casomorphin 1-5, amide, bovine;[DAla^(2,4),Tyr⁵]-beta-casomorphin 1-5, amide, bovine; [DAla²,(pCl)Phe³]-beta-casomorphin, amide, bovine; [DAla²]-beta-casomorphin1-4, amide, bovine; [DAla²]-beta-casomorphin 1-5, bovine;[DAla²]-beta-casomorphin 1-5, amide, bovine;[DAla²,Met⁵]-beta-casomorphin 1-5, bovine; [DPro²]-beta-casomorphin 1-5,amide, bovine; [DAla²]-beta-casomorphin 1-6, bovine;[DPro²]-beta-casomorphin 1-4, amide; [Des-Tyr¹]-beta-casomorphin,bovine; [DAla^(2,4),Tyr⁵]-beta-casomorphin 1-5, amide, bovine; [DAla²,(pCl)Phe³]-beta-casomorphin, amide, bovine; [DAla²]-beta-casomorphin1-4, amide, bovine; [DAla²]-beta-casomorphin 1-5, bovine;[DAla²]-beta-casomorphin 1-5, amide, bovine; [DAla²,Met⁵]-beta-casomorphin 1-5, bovine; [DPro²]-beta-casomorphin 1-5, amide,bovine; [DAla²]-beta-casomorphin 1-6, bovine; [DPro²]-beta-casomorphin1-4, amide; [Des-Tyrl]-beta-casomorphin, bovine; and[Val³]-beta-casomorphin 1-4, amide, bovine.

[0118] Chemotactic peptides including, but not limited to, defensin 1(human) HNP-1 (human neutrophil peptide-1); and N-formyl-Met-Leu-Phe.

[0119] Cholecystokinin (CCK) peptides including, but not limited to,caerulein; cholecystokinin; cholecystokinin-pancreozymin; CCK-33, human;cholecystokinin octapeptide 1-4 (non-sulfated) (CCK 26-29, unsulfated);cholecystokinin octapeptide (CCK 26-33); cholecystokinin octapeptide(non-sulfated) (CCK 26-33, unsulfated); cholecystokinin heptapeptide(CCK 27-33); cholecystokinin tetrapeptide (CCK 30-33); CCK-33, porcine;CR 1 409, cholecystokinin antagonist; CCK flanking peptide (unsulfated);N-acetyl cholecystokinin, CCK 26-30, sulfated; N-acetyl cholecystokinin,CCK 26-31, sulfated; N-acetyl cholecystokinin, CCK 26-31, non-sulfated;prepro CCK fragment V-9-M; and proglumide.

[0120] Colony-stimulating factor peptides including, but not limited to,colony-stimulating factor (CSF); GMCSF; MCSF; and G-CSF.

[0121] Corticortropin releasing factor (CRF) peptides including, but notlimited to, astressin; alpha-helical CRF 12-41; biotinyl-CRF, ovine;biotinyl-CRF, human, rat; CRF, bovine; CRF, human, rat; CRF, ovine; CRF,porcine; [Cys²¹]-CRF, human, rat; CRF antagonist (alpha-helical CRF9-41); CRF 6-33, human, rat; [DPro⁵]-CRF, human, rat; [D-Phe¹²,Nle^(21,38)]-CRF 12-41, human, rat; eosinophilotactic peptide;[Met(0)²¹]-CRF, ovine; [Nle²¹,Tyr³²]-CRF, ovine; prepro CRF 125-151,human; sauvagine, frog; [Tyr⁰]-CRF, human, rat; [Tyr⁰]-CRF, ovine;[Tyr⁰]-CRF 34-41, ovine; [Tyr⁰]-urocortin; urocortin amide, human;urocortin, rat; urotensin I (Catostomus commersoni); urotensin II; andurotensin II (Rana ridibunda).

[0122] Cortistatin peptides including, but not limited to, cortistatin29; cortistatin 29 (1-13); [Tyr⁰]-cortistatin 29; pro-cortistatin 28-47;and pro-cortistatin 51-81.

[0123] Cytokine peptides including, but not limited to, tumor necrosisfactor; and tumor necrosis factor-β (TNF-β).

[0124] Dermorphin peptides including, but not limited to, dermorphin anddermorphin analog 1-4.

[0125] Dynorphin peptides including, but not limited to, big dynorphin(prodynorphin 209-240), porcine; biotinyl-dynorphin A(biotinyl-prodynorphin 209-225); [DAla², DArg⁶]-dynorphin A 1-13,porcine; [D-Ala²]-dynorphin A, porcine; [D-Ala²]-dynorphin A amide,porcine; [D-Ala²]-dynorphin A 1-13, amide, porcine; [D-Ala²]-dynorphin A1-9, porcine; [DArg⁶]-dynorphin A 1-13, porcine; [DArg⁸]-dynorphin A1-13, porcine; [Des-Tyr¹]-dynorphin A 1-8; [D-Pro¹⁰]-dynorphin A 1-11,porcine; dynorphin A amide, porcine; dynorphin A 1-6, porcine; dynorphinA 1-7, porcine; dynorphin A 1-8, porcine; dynorphin A 1-9, porcine;dynorphin A 1-10, porcine; dynorphin A 1-10 amide, porcine; dynorphin A1-11, porcine; dynorphin A 1-12, porcine; dynorphin A 1-13, porcine;dynorphin A 1-13 amide, porcine; DAKLI (dynorphin A-analogue kappaligand); DAKLI-biotin ([Arg^(11,13)]-dynorphin A(1-13)-Gly-NH(CH2)5NH-biotin); dynorphin A 2-17, porcine; dynorphin2-17, amide, porcine; dynorphin A 2-12, porcine; dynorphin A 3-17,amide, porcine; dynorphin A 3-8, porcine; dynorphin A 3-13, porcine;dynorphin A 3-17, porcine; dynorphin A 7-17, porcine; dynorphin A 8-17,porcine; dynorphin A 6-17, porcine; dynorphin A 13-17, porcine;dynorphin A (prodynorphin 209-225), porcine; dynorphin B 1-9; [MeTyr¹,MeArg⁷, D-Leu⁸]-dynorphin 1-8 ethyl amide; [(nMe)Tyr¹] dynorphin A 1-13,amide, porcine; [Phe⁷]-dynorphin A 1-7, porcine; [Phe⁷]-dynorphin A 1-7,amide, porcine; and prodynorphin 228-256 (dynorphin B 29) (leumorphin),porcine.

[0126] Endorphin peptides including, but not limited to,alpha-neo-endorphin, porcine; beta-neo-endorphin; Ac-beta-endorphin,camel, bovine, ovine; Ac-beta-endorphin 1-27, camel, bovine, ovine;Ac-beta-endorphin, human; Ac-beta-endorphin 1-26, human;Ac-beta-endorphin 1-27, human; Ac-gamma-endorphin (Ac-beta-lipotropin61-77); acetyl-alpha-endorphin; alpha-endorphin (beta-lipotropin 61-76);alpha-neo-endorphin analog; alpha-neo-endorphin 1-7;[Arg⁸]-alpha-neo-endorphin 1-8; beta-endorphin (beta-lipotropin 61-91),camel, bovine, ovine; beta-endorphin 1-27, camel, bovine, ovine;beta-endorphin, equine; beta-endorphin (beta-lipotropin 61-91), human;beta-endorphin (1-5)+(16-31), human; beta-endorphin 1-26, human;beta-endorphin 1-27, human; beta-endorphin 6-31, human; beta-endorphin18-31, human; beta-endorphin, porcine; beta-endorphin, rat;beta-lipotropin 1-10, porcine; beta-lipotropin 60-65; beta-lipotropin61-64; beta-lipotropin 61-69; beta-lipotropin 88-91;biotinyl-beta-endorphin (biotinyl-beta-lipotropin 61-91);biocytin-beta-endorphin, human; gamma-endorphin (beta-lipotropin 61-77);[DAla²]-alpha-neo-endorphin 1-2, amide; [DAla²]-beta-lipotropin 61-69;[DAla²]-gamma-endorphin; [Des-Tyr¹]-beta-endorphin, human;[Des-Tyr¹]-gamma-endorphin (beta-lipotropin 62-77);[Leu⁵]-beta-endorphin, camel, bovine, ovine; [Met⁵,Lys⁶]-alpha-neo-endorphin 1-6; [Met⁵, Lys^(6,7)]-alpha-neo-endorphin1-7; and [Met⁵, Lys⁶, Arg⁷]-alpha-neo-endorphin 1-7.

[0127] Endothelin peptides including, but not limited to, endothelin-1(ET-1); endothelin-1[Biotin-Lys⁹]; endothelin-1 (1-15), human;endothelin-1 (1-15), amide, human; Ac-endothelin-1 (16-21), human;Ac-[DTrp¹⁶]-endothelin-1 (16-21), human; [Ala^(3,11)]-endothelin-1;[Dprl, Asp¹⁵]-endothelin-1; [Ala²]-endothelin-3, human;[Ala¹⁸]-endothelin-1, human; [Asn¹⁸]-endothelin-1, human;[Res-701-1]-endothelin B receptor antagonist; Suc-[Glu⁹,Ala^(11,15)]-endothelin-1 (8-21), IRL-1620; endothelin-C-terminalhexapeptide; [D-Val²²]-big endothelin-1 (16-38), human; endothelin-2(ET-2), human, canine; endothelin-3 (ET-3), human, rat, porcine, rabbit;biotinyl-endothelin-3 (biotinyl-ET-3); prepro-endothelin-1 (94-109),porcine; BQ-518; BQ-610; BQ-788; endothelium-dependent relaxationantagonist; FR139317; IRL-1038; JKC-30 1; JKC-302; PD-145065; PD 142893;sarafotoxin S6a (atractaspis engaddensis); sarafotoxin S6b (atractaspisengaddensis); sarafotoxin S6c (atractaspis engaddensis);[Lys⁴]-sarafotoxin S6c; sarafotoxin S6d; big endothelin-1, human;biotinyl-big endothelin-1, human; big endothelin-1 (1-39), porcine; bigendothelin-3 (22-41), amide, human; big endothelin-1 (22-39), rat; bigendothelin-1 (1-39), bovine; big endothelin-1 (22-39), bovine; bigendothelin-1 (19-38), human; big endothelin-1 (22-38), human; bigendothelin-2, human; big endothelin-2 (22-37), human; big endothelin-3,human; big endothelin-1, porcine; big endothelin-1 (22-39)(prepro-endothelin-1 (74-91)); big endothelin-1, rat; big endothelin-2(1-38), human; big endothelin-2 (22-38), human; big endothelin-3, rat;biotinyl-big endothelin-1, human; and [Tyr¹²³]-prepro-endothelin(110-130), amide, human.

[0128] ETa receptor antagonist peptides including, but not limited to,[BQ-123]; [BE18257B]; [BE-18257A]/[W-7338A]; [BQ-485]; FR139317;PD-151242; and TTA-386.

[0129] ETb receptor antagonist peptides including, but not limited to,[BQ-3020]; [RES-701-3]; and [IRL-1720]

[0130] Enkephalin peptides including, but not limited to, adrenorphin,free acid; amidorphin (proenkephalin A (104-129)-NH2), bovine; BAM-12P(bovine adrenal medulla dodecapeptide); BAM-22P (bovine adrenal medulladocosapeptide); benzoyl-Phe- Ala-Arg; enkephalin; [D-Ala²,D-Leu⁵]-enkephalin; [D-Ala², D-Met⁵]-enkephalin; [DAla²]-Leu-enkephalin,amide; [DAla²,Leu⁵,Arg⁶]-enkephalin; [Des-Tyr¹,DPen^(2,5)]-enkephalin;[Des-Tyr¹,DPen²,Pen⁵]-enkephalin; [Des-Tyr¹]-Leu-enkephalin;[D-Pen^(2,5)]-enkephalin; [DPen², Pen⁵]-enkephalin; enkephalinasesubstrate; [D-Pen², pCI-Phe⁴, D-Pen⁵]-enkephalin; Leu-enkephalin;Leu-enkephalin, amide; biotinyl-Leu-enkephalin; [D-Ala²]-Leu-enkephalin;[D-Ser²]-Leu-enkephalin-Thr (delta-receptor peptide) (DSLET);[D-Thr²]-Leu-enkephalin-Thr (DTLET); [Lys⁶]-Leu-enkephalin;[Met⁵,Arg⁶]-enkephalin; [Met⁵,Arg⁶]-enkephalin-Arg;[Met⁵,Arg⁶,Phe⁷]-enkephalin, amide; Met-enkephalin;biotinyl-Met-enkephalin; [D-Ala²]-Met-enkephalin;[D-Ala²]-Met-enkephalin, amide; Met-enkephalin-Arg-Phe; Met-enkephalin,amide; [Ala²]-Met-enkephalin, amide; [DMet²,Pro⁵]-enkephalin, amide;[DTrp²]-Met-enkephalin, amide, metorphinamide (adrenorphin); peptide B,bovine; 3200-Dalton adrenal peptide E, bovine; peptide F, bovine;preproenkephalin B 186-204, human; spinorphin, bovine; and thiorphan (D,L, 3-mercapto-2-benzylpropanoyl-glycine).

[0131] Fibronectin peptides including, but not limited to plateletfactor-4 (58-70), human; echistatin (Echis carinatus); E, P ,L selectinconserved region; fibronectin analog; fibronectin-binding protein;fibrinopeptide A, human; [Tyr⁰]-fibrinopeptide A, human; fibrinopeptideB, human; [Glu¹]-fibrinopeptide B, human; [Tyr¹⁵]-fibrinopeptide B,human; fibrinogen beta-chain fragment of 24-42; fibrinogen bindinginhibitor peptide; fibronectin related peptide (collagen bindingfragment); fibrinolysis inhibiting factor; FN-C/H-1 (fibronectinheparin-binding fragment); FN-C/H-V (fibronectin heparin-bindingfragment); heparin-binding peptide; laminin penta peptide, amide;Leu-Asp-Val-NH2 (LDV-NH2), human, bovine, rat, chicken; necrofibrin,human; necrofibrin, rat; and platelet membrane glycoprotein IIB peptide296-306.

[0132] Galanin peptides including, but not limited to, galanin, human;galanin 1-19, human; preprogalanin 1-30, human; preprogalanin 65-88,human; preprogalanin 89-123, human; galanin, porcine; galanin 1-16,porcine, rat; galanin, rat; biotinyl-galanin, rat; preprogalanin 28-67,rat; galanin 1-13-bradykinin 2-9, amide; M40, galanin1-13-Pro-Pro-(Ala-Leu) 2-Ala-amide; C7, galanin 1-13-spantide-amide;GMAP 1-41, amide; GMAP 16-41, amide; GMAP 25-41, amide; galantide; andentero-kassinin.

[0133] Gastrin peptides including, but not limited to, gastrin, chicken;gastric inhibitory peptide (GIP), human; gastrin I, human;biotinyl-gastrin I, human; big gastrin-1, human; gastrin releasingpeptide, human; gastrin releasing peptide 1-16, human; gastricinhibitory polypeptide (GIP), porcine; gastrin releasing peptide,porcine; biotinyl-gastrin releasing peptide, porcine; gastrin releasingpeptide 14-27, porcine, human; little gastrin, rat; pentagastrin;gastric inhibitory peptide 1-30, porcine; gastric inhibitory peptide1-30, amide, porcine; [Tyr⁰]-gastric inhibitory peptide 23-42, human;and gastric inhibitory peptide, rat.

[0134] Glucagon peptides including, but not limited to,[Des-His¹,Glu⁹]-glucagon, extendin-4, glucagon, human;biotinyl-glucagon, human; glucagon 19-29, human; glucagon 22-29, human;Des-His¹-[Glu⁹]-glucagon, amide; glucagon-like peptide 1, amide(preproglucagon 72-107, amide); glucagon-like peptide 1 (preproglucagon72-108), human; glucagon-like peptide 1 (7-36) (preproglucagon 78-107,amide); glucagon-like peptide II, rat; biotinyl-glucagon-like peptide-1(7-36) (biotinyl-preproglucagon 78-107, amide); glucagon-like peptide 2(preproglucagon 126-159), human; oxyntomodulin/glucagon 37; and valosin(peptide VQY), porcine.

[0135] Gn-RH associated peptides (GAP) including, but not limited to,Gn-RH associated peptide 25-53, human; Gn-RH associated peptide 1-24,human; Gn-RH associated peptide 1-13, human; Gn-RH associated peptide 1-13, rat; gonadotropin releasing peptide, follicular, human; [Tyr⁰]-GAP([Tyr⁰]-Gn-RH Precursor Peptide 14-69), human; and proopiomelanocortin(POMC) precursor 27-52, porcine.

[0136] Growth factor peptides including, but not limited to, cell growthfactors; epidermal growth factors; tumor growth factor; alpha-TGF;beta-TF; alpha-TGF 34-43, rat; EGF, human; acidic fibroblast growthfactor; basic fibroblast growth factor; basic fibroblast growth factor13-18; basic fibroblast growth factor 120-125; brain derived acidicfibroblast growth factor 1-1; brain derived basic fibroblast growthfactor 1-24; brain derived acidic fibroblast growth factor 102-111;[Cys(Acm^(20,31))]-epidermal growth factor 20-31; epidermal growthfactor receptor peptide 985-996; insulin-like growth factor (IGF)-I,chicken; IGF-I, rat; IGF-I, human; Des (1-3) IGF-I, human; R3 IGF-I,human; R3 IGF-I, human; long R3 IGF-I, human; adjuvant peptide analog;anorexigenic peptide; Des (1-6) IGF-II, human; R6 IGF-II, human; IGF-Ianalogue; IGF I (24-41); IGF I (57-70); IGF I (30-41); IGF II; IGF II(33-40); [Tyr⁰]-IGF II (33-40); liver cell growth factor; midkine;midkine 60-121, human; N-acetyl, alpha-TGF 34-43, methyl ester, rat;nerve growth factor (NGF), mouse; platelet-derived growth factor;platelet-derived growth factor antagonist; transforming growthfactor-alpha, human; and transforming growth factor-I, rat.

[0137] Growth hormone peptides including, but not limited to, growthhormone (hGH), human; growth hormone 1-43, human; growth hormone 6-13,human; growth hormone releasing factor, human; growth hormone releasingfactor, bovine; growth hormone releasing factor, porcine; growth hormonereleasing factor 1-29, amide, rat; growth hormone pro-releasing factor,human; biotinyl-growth hormone releasing factor, human; growth hormonereleasing factor 1-29, amide, human; [D-Ala²] -growth hormone releasingfactor 1-29, amide, human; [N-Ac-Tyr¹, D-Arg²]-GRF 1-29, amide; [His¹,Nle²⁷]-growth hormone releasing factor 1-32, amide; growth hormonereleasing factor 1-37, human; growth hormone releasing factor 1-40,human; growth hormone releasing factor 1-40, amide, human; growthhormone releasing factor 30-44, amide, human; growth hormone releasingfactor, mouse; growth hormone releasing factor, ovine; growth hormonereleasing factor, rat; biotinyl- growth hormone releasing factor, rat;GHRP-6 ([His¹, Lys⁶]-GHRP); hexarelin (growth hormone releasinghexapeptide); and [D-Lys³]-GHRP-6.

[0138] GTP-binding protein fragment peptides including, but not limitedto, [Arg⁸]-GTP-binding protein fragment, Gs alpha; GTP-binding proteinfragment, G beta; GTP-binding protein fragment, GAlpha; GTP-bindingprotein fragment, Go Alpha; GTP-binding protein fragment, Gs Alpha; andGTP-binding protein fragment, G Alpha i2.

[0139] Guanylin peptides including, but not limited to, guanylin, human;guanylin, rat; and uroguanylin.

[0140] Inhibin peptides including, but not limited to, inhibin, bovine;inhibin, alpha-subunit 1-32, human; [Tyr⁰]-inhibin, alpha-subunit 1-32,human; seminal plasma inhibin-like peptide, human; [Tyr⁰]-seminal plasmainhibin-like peptide, human; inhibin, alpha-subunit 1-32, porcine; and[Tyr⁰]-inhibin, alpha-subunit 1-32, porcine.

[0141] Insulin peptides including, but not limited to, insulin, human;insulin, porcine; IGF-I, human; insulin-like growth factor II (69-84);pro-insulin-like growth factor II (68-102), human; pro-insulin-likegrowth factor II (105-128), human; [Asp^(B28)]-insulin, human;[Lys^(B28)]-insulin, human; [Leu^(B28)]-insulin, human;[Val^(B28)]-insulin, human; [Ala^(B28)]-insulin, human;[Asp^(B28),Pro^(B29)]-insulin, human; [Lys^(B28), Pro^(B29)]-insulin,human; [Leu^(B28), Pro^(B29)]-insulin, human; [Val^(B28)S,Pro^(B29)]-insulin, human; and [Ala^(B28), Pro^(B29)]-insulin, human,B22-B30 insulin, human; B23-B30 insulin, human; B25-B30 insulin, human;B26-B30 insulin, human; B27-B30 insulin, human; B29-B30 insulin, human;the A chain of human insulin, and the B chain of human insulin.

[0142] Interleukin peptides including, but not limited to, interleukin-1beta 165-181, rat; and interleukin-8 (IL-8, CINC/gro), rat.

[0143] Laminin peptides including, but not limited to, laminin; alphal(I)-CB3 435-438, rat; and laminin binding inhibitor.

[0144] Leptin peptides including, but not limited to, leptin 93-105,human; leptin 22-56, rat; Tyr-leptin 26-39, human; and leptin 116-130,amide, mouse.

[0145] Leucokinin peptides including, but not limited to,leucomyosuppressin (LMS); leucopyrokinin (LPK); leucokinin I; leucokininII; leucokinin III; leucokinin IV; leucokinin VI; leucokinin VII; andleucokinin VIII.

[0146] Luteinizing hormone-releasing hormone peptides including, but notlimited to, antide; Gn-RH II, chicken; luteinizing hormone-releasinghormone (LH-RH) (GnRH); biotinyl-LH-RH; cetrorelix (D-20761);[D-Ala⁶]-LH-RH; [Gln⁸]-LH-RH (Chicken LH-RH); [DLeu⁶, Val⁷]LH-RH 1-9,ethyl amide; [D-Lys⁶]-LH-RH; [D-Phe², Pro³, D-Phe⁶]-LH-RH; [DPhe²,DAla⁶] LH-RH; [Des-Gly¹⁰]-LH-RH, ethyl amide; [D-Ala⁶, Des-Gly¹⁰]-LH-RH,ethyl amide; [DTrp⁶]-LH-RH, ethyl amide; [D-Trp⁶, Des-Gly¹⁰]-LH-RH,ethyl amide (Deslorelin); [DSer(But)⁶, Des-Gly¹⁰]-LH-RH, ethyl amide;ethyl amide; leuprolide; LH-RH 4-10; LH-RH 7-10; LH-RH, free acid;LH-RH, lamprey; LH-RH, salmon; [Lys⁸]-LH-RH; [Trp⁷,Leu⁸] LH-RH, freeacid; and [(t-Bu)DSer⁶, (Aza)Gly¹⁰]-LH-RH.

[0147] Mastoparan peptides including, but not limited to, mastoparan;mas7; mas8; mas17; and mastoparan X.

[0148] Mast cell degranulating peptides including, but not limited to,mast cell degranulating peptide HR-1; and mast cell degranulatingpeptide HR-2.

[0149] Melanocyte stimulating hormone (MSH) peptides including, but notlimited to, [Ac-Cys⁴,DPhe⁷,Cys¹⁰] alpha-MSH 4-13, amide;alpha-melanocyte stimulating hormone; alpha-MSH, free acid; beta-MSH,porcine; biotinyl-alpha-melanocyte stimulating hormone; biotinyl-[Nle⁴,D-Phe⁷] alpha-melanocyte stimulating hormone; [Des-Acetyl]-alpha-MSH;[DPhe⁷]-alpha-MSH, amide; gamma-1-MSH, amide; [Lys⁰]-gamma-1-MSH, amide;MSH release inhibiting factor, amide; [Nle⁴]-alpha-MSH, amide; [Nle⁴,D-Phe⁷]-alpha-MSH; N-Acetyl, [Nle⁴,DPhe⁷] alpha-MSH 4-10, amide;beta-MSH, human; and gamma-MSH.

[0150] Morphiceptin peptides including, but not limited to, morphiceptin(beta-casomorphin 1-4 amide); [D-Pro⁴]-morphiceptin; and[N-MePhe³,D-Pro⁴]-morphiceptin.

[0151] Motilin peptides including, but not limited to, motilin, canine;motilin, porcine; biotinyl-motilin, porcine; and [Leu¹³]-motilin,porcine.

[0152] Neuro-peptides including, but not limited to, Ac-Asp-Glu;achatina cardio-excitatory peptide-1 (ACEP-1) (Achatina fulica);adipokinetic hormone (AKH) (Locust); adipokinetic hormone (Heliothis zeaand Manduca sexta); alytesin; Tabanus atratus adipokinetic hormone(Taa-AKH); adipokinetic hormone II (Locusta migratoria); adipokinetichormone II (Schistocera gregaria); adipokinetic hormone III (AKH-3);adipokinetic hormone G (AKH-G) (Gryllus bimaculatus); allatotropin (AT)(Manduca sexta); allatotropin 6-13 (Manduca sexta); APGW amide (Lymnaeastagnalis); buccalin; cerebellin; [Des-Ser¹]-cerebellin; corazonin(American Cockroach Periplaneta americana); crustacean cardioactivepeptide (CCAP); crustacean erythrophore; DF2 (Procambarus clarkii);diazepam-binding inhibitor fragment, human; diazepam binding inhibitorfragment (ODN); eledoisin related peptide; FMRF amide (molluscancardioexcitatory neuro-peptide); Gly-Pro-Glu (GPE), human; granuliberinR; head activator neuropeptide; [His⁷]-corazonin; stick insecthypertrehalosaemic factor II; Tabanus atratus hypotrehalosemic hormone(Taa-HoTH); isoguvacine hydrochloride; bicuculline methiodide;piperidine-4-sulphonic acid; joining peptide of proopiomelanocortin(POMC), bovine; joining peptide, rat; KSAYMRF amide (P. redivivus);kassinin; kinetensin; levitide; litorin; LUQ 81-91 (Aplysiacalifornica); LUQ 83-91 (Aplysia californica); myoactive peptide I(Periplanetin CC-1) (Neuro-hormone D); myoactive peptide II(Periplanetin CC-2); myomodulin; neuron specific peptide; neuronspecific enolase 404-443, rat; neuropeptide FF; neuropeptide K, porcine;NEI (prepro-MCH 131-143) neuropeptide, rat; NGE (prepro-MCH 110-128)neuropeptide, rat; NF1 (Procambarus clarkii); PBAN-1 (Bombyx mori);Hez-PBAN (Heliothis zea); SCPB (cardioactive peptide from aplysia);secretoneurin, rat; uperolein; urechistachykinin I; urechistachykininII; xenopsin-related peptide I; xenopsin-related peptide II; pedalpeptide (Pep), aplysia; peptide F1, lobster; phyllomedusin; polistesmastoparan; proctolin; ranatensin; Ro I (Lubber Grasshopper, Romaleamicroptera); Ro II (Lubber Grasshopper, Romalea microptera); SALMF amide1 (S1); SALMF amide 2 (S2); and SCPA.

[0153] Neuropeptide Y (NPY) peptides including, but not limited to,[Leu³¹,Pro³⁴]-neuropeptide Y, human; neuropeptide F (Moniezia expansa);B1BP3226 NPY antagonist; Bis (31/31′) {[Cys³¹, Trp³², Nva³⁴] NPY 31-36};neuropeptide Y, human, rat; neuropeptide Y1-24 amide, human;biotinyl-neuropeptide Y; [D-Tyr^(27,36), D-Thr³²]-NPY 27-36; Des 10-17(cyclo 7-21) [Cys^(7,21), Pro³⁴]-NPY; C2-NPY; [Leu³¹, Pro³⁴]neuropeptide Y, human; neuropeptide Y, free acid, human; neuropeptide Y,free acid, porcine; prepro NPY 68-97, human; N-acetyl-[Leu²⁸, Leu³¹] NPY24-36; neuropeptide Y, porcine; [D-Trp³²]-neuropeptide Y, porcine;[D-Trp³²] NPY 1-36, human; [Leu¹⁷,DTrp³²] neuropeptide Y, human; [Leu³¹,Pro³⁴]-NPY, porcine; NPY 2-36, porcine; NPY 3-36, human; NPY 3-36,porcine; NPY 13-36, human; NPY 13-36, porcine; NPY 16-36. porcine; NPY18-36, porcine; NPY 20-36; NFY 22-36; NPY 26-36; [Pro³⁴]-NPY 1-36,human; [Pro³⁴]-neuropeptide Y, porcine; PYX-1; PYX-2; T4-[NPY(33-36)]4;and Tyr(OMe)²¹]-neuropeptide Y, human.

[0154] Neurotropic factor peptides including, but not limited to, glialderived neurotropic factor (GDNF); brain derived neurotropic factor(BDNF); and ciliary neurotropic factor (CNTF).

[0155] Orexin peptides including, but not limited to, orexin A; orexinB, human; orexin B, rat, mouse.

[0156] Opioid peptides including, but not limited to, alpha-caseinfragment 90-95; BAM-18P; casomokinin L; casoxin D; crystalline; DALDA;dermenkephalin (deltorphin) (Phylomedusa sauvagei); [D-Ala²]-deltorphinI; [D-Ala²]-deltorphin II; endomorphin-1; endomorphin-2; kyotorphin;[DArg²]-kyotorphin; morphin tolerance peptide; morphine modulatingpeptide, C-terminal fragment; morphine modulating neuropeptide(A-18-F-NH2); nociceptin [orphanin FQ] (ORLI agonist); TIPP; Tyr-MIF-1;Tyr-W-MIF-1; valorphin; LW-hemorphin-6, human; Leu-valorphin-Arg; andZ-Pro-D-Leu.

[0157] Oxytocin peptides including, but not limited to, [Asu⁶]-oxytocin;oxytocin; biotinyl-oxytocin; [Thr⁴, Gly⁷]-oxytocin; and tocinoic acid([Ile³]-pressinoic acid).

[0158] PACAP (pituitary adenylating cyclase activating peptide) peptidesincluding, but not limited to, PACAP 1-27, human, ovine, rat; PACAP(1-27)-Gly-Lys-Arg-NH2, human; [Des-Gln⁶]-PACAP 6-27, human, ovine, rat;PACAP38, frog; PACAP27-NH2, human, ovine, rat; biotinyl-PACAP27-NH2,human, ovine, rat; PACAP 6-27, human, ovine, rat; PACAP38, human, ovine,rat; biotinyl-PACAP38, human, ovine, rat; PACAP 6-38, human, ovine, rat;PACAP27-NH2, human, ovine, rat; biotinyl-PACAP27-NH2, human, ovine, rat;PACAP 6-27, human, ovine, rat; PACAP38, human, ovine, rat;biotinyl-PACAP38, human, ovine, rat; PACAP 6-38, human, ovine, rat;PACAP38 16-38, human, ovine, rat; PACAP38 31-38, human, ovine, rat;PACAP38 31-38, human, ovine, rat; PACAP-related peptide (PRP), human;and PACAP-related peptide (PRP), rat.

[0159] Pancreastatin peptides including, but not limited to,chromostatin, bovine; pancreastatin (hPST-52) (chromogranin A 250-301,amide); pancreastatin 24-52 (hPST-29), human; chromogranin A 286-301,amide, human; pancreastatin, porcine; biotinyl-pancreastatin, porcine;[Nle⁸]-pancreastatin, porcine; [Tyr⁰,Nle⁸]-pancreastatin, porcine;[Tyr⁰]-pancreastatin, porcine; parastatin 1-19 (chromogranin A 347-365),porcine; pancreastatin (chromogranin A 264-314-amide, rat;biotinyl-pancreastatin (biotinyl-chromogranin A 264-314-amide;[Tyr⁰]-pancreastatin, rat; pancreastatin 26-51, rat; and pancreastatin33-49, porcine.

[0160] Pancreatic polypeptides including, but not limited to, pancreaticpolypeptide, avian; pancreatic polypeptide, human; C-fragment pancreaticpolypeptide acid, human; C-fragment pancreatic polypeptide amide, human;pancreatic polypeptide (Rana temporaria); pancreatic polypeptide, rat;and pancreatic polypeptide, salmon.

[0161] Parathyroid hormone peptides including, but not limited to,[Asp⁷⁶]-parathyroid hormone 39-84, human; [Asp⁷⁶]-parathyroid hormone53-84, human; [Asn⁷⁶]-parathyroid hormone 1-84, hormone;[Asn⁷⁶]-parathyroid hormone 64-84, human; [Asn⁸, Leu¹⁸]-parathyroidhormone 1-34, human; [Cys^(5,28)]-parathyroid hormone 1-34, human;hypercalcemia malignancy factor 1-40; [Leu¹⁸]-parathyroid hormone 1-34,human; [Lys(biotinyl)¹³, Nle^(8,18), Tyr³⁴]-parathyroid hormone 1-34amide; [Nle^(8,18), Tyr³⁴]-parathyroid hormone 1-34 amide; [Nle^(8,18),Tyr³⁴]-parathyroid hormone 3-34 amide, bovine; [Nle^(8,18),Tyr³⁴]-parathyroid hormone 1-34, human; [Nle^(8,18), Tyr³⁴]-parathyroidhormone 1-34 amide, human; [Nle^(8,18), Tyr³⁴]-parathyroid hormone 3-34amide, human; [Nle^(8,18), Tyr³⁴]- parathyroid hormone 7-34 amide,bovine; [Nle^(8,21), Tyr³⁴]-parathyroid hormone 1-34 amide, rat;parathyroid hormone 44-68, human; parathyroid hormone 1-34, bovine;parathyroid hormone 3-34, bovine; parathyroid hormone 1-31 amide, human;parathyroid hormone 1-34, human; parathyroid hormone 13-34, human;parathyroid hormone 1-34, rat; parathyroid hormone 1-38, human;parathyroid hormone 1-44, human; parathyroid hormone 28-48, human;parathyroid hormone 39-68, human; parathyroid hormone 39-84, human;parathyroid hormone 53-84, human; parathyroid hormone 69-84, human;parathyroid hormone 70-84, human; [Pro³⁴]-peptide YY (PYY), human;[Tyr⁰]-hypercalcemia malignancy factor 1-40; [Tyr⁰]-parathyroid hormone1-44, human; [Tyr⁰]-parathyroid hormone 1-34, human; [Tyr¹]-parathyroidhormone 1-34, human; [Tyr²⁷]-parathyroid hormone 27-48, human;[Tyr³⁴]-parathyroid hormone 7-34 amide, bovine; [Tyr⁴³]-parathyroidhormone 43-68, human; [Tyr⁵², Asn⁷⁶]-parathyroid hormone 52-84, human;and [Tyr⁶³]-parathyroid hormone 63-84, human.

[0162] Parathyroid hormone (PTH)-related peptides including, but notlimited to, PTHrP ([Tyr³⁶]-PTHrP 1-36 amide), chicken; hHCF-(1-34)-NH2(humoral hypercalcemic factor), human; PTH-related protein 1-34, human;biotinyl-PTH-related protein 1-34, human; [Tyr⁰]-PTH-related protein1-34, human; [Tyr³⁴]-PTH-related protein 1-34 amide, human; PTH-relatedprotein 1-37, human; PTH-related protein 7-34 amide, human; PTH-relatedprotein 38-64 amide, human; PTH-related protein 67-86 amide, human;PTH-related protein 107-111, human, rat, mouse; PTH-related protein107-111 free acid; PTH-related protein 107-138, human; and PTH-relatedprotein 109-111, human.

[0163] Peptide T peptides including, but not limited to, peptide T;[D-Alal]-peptide T; and [D-Alal]-peptide T amide.

[0164] Prolactin-releasing peptides including, but not limited to,prolactin-releasing peptide 31, human; prolactin-releasing peptide 20,human; prolactin-releasing peptide 31, rat; prolactin-releasing peptide20, rat; prolactin-releasing peptide 31, bovine; and prolactin-releasingpeptide 20, bovine.

[0165] Peptide YY (PYY) peptides including, but not limited to, PYY,human; PYY 3-36, human; biotinyl-PYY, human; PYY, porcine, rat; and[Leu³¹, Pro³⁴]-PYY, human.

[0166] Renin substrate peptides including, but not limited to, acetyl,angiotensinogen 1-14, human; angiotensinogen 1-14, porcine; reninsubstrate tetradecapeptide, rat; [Cys⁸]-renin substratetetradecapeptide, rat; [Leu⁸]-renin substrate tetradecapeptide, rat; and[Val⁸]-renin substrate tetradecapeptide, rat.

[0167] Secretin peptides including, but not limited to, secretin,canine; secretin, chicken; secretin, human; biotinyl-secretin, human;secretin, porcine; and secretin, rat.

[0168] Somatostatin (GIF) peptides including, but not limited to,BIM-23027; biotinyl-somatostatin; biotinylated cortistatin 17, human;cortistatin 14, rat; cortistatin 17, human; [Tyr⁰]-cortistatin 17,human; cortistatin 29, rat; [D-Trp⁸]-somatostatin;[DTrp⁸,DCys¹⁴]-somatostatin; [DTrp⁸,Tyr¹¹]-somatostatin;[D-Trp¹¹]-somatostatin; NTB (Naltriben); [Nle⁸]-somatostatin 1-28;octreotide (SMS 201-995); prosomatostatin 1-32, porcine;[Tyr⁰]-somatostatin; [Tyr¹]-somatostatin; [Tyr¹]-somatostatin 28 (1-14);[Tyr¹¹]-somatostatin; [Tyr⁰, D-Trp⁸]-somatostatin; somatostatin;somatostatin antagonist; somatostatin-25; somatostatin-28; somatostatin28 (1-12); biotinyl-somatostatin-28; [Tyr⁰]-somatostatin-28; [Leu⁸,D-Trp²², Tyr²⁵]-somatostatin-28; biotinyl-[Leu⁸, D-Trp²²,Tyr²⁵]-somatostatin-28; somatostatin-28 (1-14); and somatostatin analog,RC-160.

[0169] Substance P peptides including, but not limited to, G proteinantagonist-2; Ac-[Arg⁶, Sar⁹, Met(02)¹¹]-substance P 6-11;[Arg³]-substance P; Ac-Trp-3,5-bis(trifluoromethyl) benzyl ester;Ac-[Arg⁶, Sar⁹, Met(O2)¹¹]-substance P 6-11; [D-Ala⁴]-substance P 4-11;[Tyr⁶, D-Phe⁷, D-His⁹]-substance P 6-11 (sendide); biotinyl-substance P;biotinyl-NTE[Arg³]-substance P; [Tyr⁸]-substance P; [Sar⁹,Met(O2)¹¹]-substance P; [D-Pro², D-Trp^(7,9)]-substance P; [D-Pro⁴,0-Trp^(7,9)]-substance P 4-11; substance P 4-11;[DTrp^(2,7,9)]-substance P; [(Dehydro)Pro^(2,4), Pro⁹]-substance P;[Dehydro-Pro⁴]-substance P 4-11; [Glp⁵,(Me)Phe⁸,Sar⁹]-substance P 5-11;[Glp⁵,Sar⁹]-substance P 5-11; [Glp⁵]-substance P 5-11; hepta-substance P(substance P 5-11); hexa-substance P(substance P 6-11);[MePhe⁸,Sar⁹]-substance P; [Nle¹¹]-substance P; Octa-substanceP(substance P 4-11); [pGlu¹]-hexa-substance P ([pGlu⁶]-substance P6-11); [pGlu⁶, D-Pro⁹]-substance P 6-11; [(pNO2)Phe⁷Nle¹¹]-substance P;penta-substance P (substance P 7-11); [Pro⁹]-substance P; GR73632,substance P 7-11; [Sar⁴]-substance P 4-11; [Sar⁹]-substance P; septide([pGlu⁶, Pro⁹]-substance P 6-11); spantide I; spantide II; substance P;substance P, cod; substance P, trout; substance P antagonist; substanceP-Gly-Lys-Arg; substance P 1-4; substance P 1-6; substance P 1-7;substance P 1-9; deca-substance P (substance P 2-11); nona-substance P(substance P 3-11); substance P tetrapeptide (substance P 8-11);substance P tripeptide (substance P 9-11); substance P, free acid;substance P methyl ester; and [Tyr⁸,Nle11] substance P.

[0170] Tachykinin peptides including, but not limited to, [Ala⁵,beta-Ala⁸] neurokinin A 4-10; eledoisin; locustatachykinin I (Lom-TK-I)(Locusta migratoria); locustatachykinin II (Lom-TK-II) (Locustamigratoria); neurokinin A 4-10; neurokinin A (neuromedin L, substanceK); neurokinin A, cod and trout; biotinyl-neurokinin A(biotinyl-neuromedin L, biotinyl-substance K); [Tyr⁰]-neurokinin A;[Tyr⁶]-substance K; FR64349; [Lys³,Gly⁸-(R)-gamma-lactam-Leu⁹]-neurokinin A 3-10; GR83074; GR87389;GR94800; [Beta-Ala⁸]-neurokinin A 4-10; [Nle¹⁰]-neurokinin A 4-10;[Trp⁷, beta-Ala⁸]-neurokinin A 4-10; neurokinin B (neuromedin K);biotinyl-neurokinin B (biotinyl-neuromedin K); [MePhe⁷]-neurokinin B;[Pro⁷]-neurokinin B; [Tyr⁰]-neurokinin B; neuromedin B, porcine;biotinyl-neuromedin B, porcine; neuromedin B-30, porcine; neuromedinB-32, porcine; neuromedin B receptor antagonist; neuromedin C, porcine;neuromedin N, porcine; neuromedin (U-8), porcine; neuromedin (U-25),porcine; neuromedin U, rat; neuropeptide-gamma (gamma-preprotachykinin72-92); PG-KII; phyllolitorin; [Leu⁸]-phyllolitorin (Phyllomedusasauvagei); physalaemin; physalaemin 1-11; scyliorhinin II, amide,dogfish; senktide, selective neurokinin B receptor peptide;[Ser²]-neuromedin C; beta-preprotachykinin 69-91, human;beta-preprotachykinin 111-129, human; tachyplesin I; xenopsin; andxenopsin 25 (xenin 25), human.

[0171] Thyrotropin-releasing hormone (TRH) peptides including, but notlimited to, biotinyl-thyrotropin-releasing hormone; [Glu¹]-TRH;His-Pro-diketopiperazine; [3-Me-His²]-TRH; pGlu-Gln-Pro-amide; pGlu-His;[Phe²]-TRH; prepro TRH 53-74; prepro TRH 83-106; prepro-TRH 160-169(Ps4, TRH-potentiating peptide); prepro-TRH 178-199;thyrotropin-releasing hormone (TRH); TRH, free acid; TRH-SH Pro; and TRHprecursor peptide.

[0172] Toxin peptides including, but not limited to, omega-agatoxin TK;agelenin, (spider, Agelena opulenta); apamin (honeybee, Apis mellifera);calcicudine (CaC) (green mamba, Dedroaspis angusticeps); calciseptine(black mamba, Dendroaspis polylepis polylepis); charybdotoxin (ChTX)(scorpion, Leiurus quinquestriatus var. hebraeus); chlorotoxin;conotoxin GI (marine snail, Conus geographus); conotoxin GS (marinesnail, Conus geographus); conotoxin MI (Marine Conus magus);alpha-conotoxin EI, Conus ermineus; alpha-conotoxin SIA; alpha-conotoxinIm1; alpha-conotoxin SI (cone snail, Conus striatus); micro-conotoxinGIIIB (marine snail, Conus geographus); omega-conotoxin GVIA (marinesnail, Conus geographus); omega-conotoxin MVIIA (Conus magus);omega-conotoxin MVIIC (Conus magus); omega-conotoxin SVIB (cone snail,Conus striatus); endotoxin inhibitor; geographutoxin I (GTX-I)(μ-Conotoxin GIIIA); iberiotoxin (IbTX) (scorpion, Buthus tamulus);kaliotoxin 1-37; kaliotoxin (scorpion, Androct-onus mauretanicusmauretanicus); mast cell-degranulating peptide (MCD-peptide, peptide401); margatoxin (MgTX) (scorpion, Centruriodes Margaritatus);neurotoxin NSTX-3 (pupua new guinean spider, Nephilia maculata); PLTX-II(spider, Plectreurys tristes); seyllatoxin (leiurotoxin I); andstichodactyla toxin (ShK).

[0173] Vasoactive intestinal peptides (VIP/PHI) including, but notlimited to, VIP, human, porcine, rat, ovine; VIP-Gly-Lys-Arg-NH2;biotinyl-PHI (biotinyl-PHI-27), porcine; [Glp¹⁶] VIP 16-28, porcine; PHI(PHI-27), porcine; PHI (PHI-27), rat; PHM-27 (PHI), human; prepro VIP81-122, human; preproVIP/PHM 111-122; prepro VIP/PHM 156-170;biotinyl-PHM-27 (biotinyl-PHI), human; vasoactive intestinal contractor(endothelin-beta); vasoactive intestinal octacosa-peptide, chicken;vasoactive intestinal peptide, guinea pig; biotinyl-VIP, human, porcine,rat; vasoactive intestinal peptide 1-12, human, porcine, rat; vasoactiveintestinal peptide 10-28, human, porcine, rat; vasoactive intestinalpeptide 11-28, human, porcine, rat, ovine; vasoactive intestinal peptide(cod, Gadus morhua); vasoactive intestinal peptide 6-28; vasoactiveintestinal peptide antagonist; vasoactive intestinal peptide antagonist([Ac-Tyr¹, D-Phe²]-GHRF 1-29 amide); vasoactive intestinal peptidereceptor antagonist (4-Cl-D-Phe⁶, Leu¹⁷]-VIP); and vasoactive intestinalpeptide receptor binding inhibitor, L-8-K.

[0174] Vasopressin (ADH) peptides including, but not limited to,vasopressin; [Asu^(1,6),Arg⁸]-vasopressin; vasotocin;[Asu^(1,6),Arg⁸]-vasotocin; [Lys⁸]-vasopressin; pressinoic acid;[Arg⁸]-desamino vasopressin desglycinamide; [Arg⁸]-vasopressin (AVP);[Arg⁸]-vasopressin desglycinamide; biotinyl-[Arg⁸]-vasopressin(biotinyl-AVP); [D-Arg⁸]-vasopressin; desamino-[Arg⁸]-vasopressin;desamino-[D-Arg⁸]-vasopressin (DDAVP);[deamino-[D-3-(3′-pyridyl-Ala)]-[Arg⁸]-vasopressin;[1-(beta-Mercapto-beta, beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine]-[Arg⁸]-vasopressin; vasopressin metaboliteneuropeptide [pGlu⁴, Cys⁶]; vasopressin metabolite neuropeptide [pGlu⁴,Cys⁶]; [Lys⁸]-deamino vasopressin desglycinamide; [Lys⁸]-vasopressin;[Mpr¹,Val⁴,DArg⁸]-vasopressin; [Phe², Ile³, Orn⁸]-vasopressin ([Phe²,Orn⁸]-vasotocin); [Arg⁸]-vasotocin; and [d(CH2)5, Tyr(Me)²,Orn⁸]-vasotocin.

[0175] Virus related peptides including, but not limited to, fluorogenichuman CMV protease substrate; HCV core protein 59-68; HCV NS4A protein18-40 (JT strain); HCV NS4A protein 21-34 (JT strain); hepatitis B virusreceptor binding fragment; hepatitus B virus pre-S region 120-145;[Ala¹²⁷]-hepatitus B virus pre-S region 120-131; herpes virus inhibitor2; HIV envelope protein fragment 254-274; HIV gag fragment 129-135; HIVsubstrate; P 18 peptide; peptide T; [3,5 diiodo-Tyr⁷] peptide T; R15SKHIV-1 inhibitory peptide; T20; T21; V3 decapeptide P 18-110; and virusreplication inhibiting peptide.

[0176] While certain analogs, fragments, and/or analog fragments of thevarious polypeptides have been described above, it is to be understoodthat other analogs, fragments, and/or analog fragments that retain allor some of the activity of the particular polypeptide may also be usefulin embodiments of the present invention. Analogs may be obtained byvarious means, as will be understood by those skilled in the art. Forexample, certain amino acids may be substituted for other amino acids ina polypeptide without appreciable loss of interactive binding capacitywith structures such as, for example, antigen-binding regions ofantibodies or binding sites on substrate molecules. As the interactivecapacity and nature of a polypeptide drug defines its biologicalfunctional activity, certain amino acid sequence substitutions can bemade in the amino acid sequence and nevertheless remain a polypeptidewith like properties.

[0177] In making such substitutions, the hydropathic index of aminoacids may be considered. The importance of the hydropathic amino acidindex in conferring interactive biologic function on a polypeptide isgenerally understood in the art. It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant polypeptide, which in turn defines theinteraction of the polypeptide with other molecules, for example,enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics as follows: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). As will be understood by those skilled in the art, certain aminoacids may be substituted by other amino acids having a similarhydropathic index or score and still result in a polypeptide withsimilar biological activity, i.e., still obtain a biologicalfunctionally equivalent polypeptide. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ofeach other is preferred, those which are within ±1 of each other areparticularly preferred, and those within ±0.5 of each other are evenmore particularly preferred.

[0178] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101 provides that the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with a biological property of theprotein. As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1);seine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4). As is understood by those skilled in the art, an amino acid canbe substituted for another having a similar hydrophilicity value andstill obtain a biologically equivalent, and in particular, animmunologically equivalent polypeptide. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ofeach other is preferred, those which are within ±1 of each other areparticularly preferred, and those within ±0.5 of each other are evenmore particularly preferred.

[0179] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions (i.e., amino acids that maybe interchanged without significantly altering the biological activityof the polypeptide) that take various of the foregoing characteristicsinto consideration are well known to those of skill in the art andinclude, for example: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

[0180] In embodiments of the present invention, a substantiallymonodispersed mixture of drug-oligomer conjugates is provided.Preferably, at least about 96, 97, 98 or 99 percent of the conjugates inthe mixture have the same molecular weight. More preferably, the mixtureis a monodispersed mixture. Even more preferably, the mixture is asubstantially purely monodispersed mixture of drug-oligomer conjugates.Still more preferably, at least about 96, 97, 98 or 99 percent of theconjugates in the mixture have the same molecular weight and the samemolecular structure. Most preferably, the mixture is a purelymonodispersed mixture.

[0181] The oligomer may be various oligomers comprising a polyalkyleneglycol moiety as will be understood by those skilled in the art.Preferably, the polyalkylene glycol moiety has at least 2, 3, or 4polyalkylene glycol subunits. More preferably, the polyalkylene glycolmoiety has at least 5 or 6 polyalkylene glycol subunits. Mo stpreferably, the polyalkylene glycol moiety of the oligomer has at least7 polyalkylene glycol subunits. The polyalkylene glycol moiety of theoligomer is preferably a lower alkyl polyalkylene glycol moiety such asa polyethylene glycol moiety, a polypropylene glycol moiety, or apolybutylene glycol moiety. When the polyalkylene moiety is apolypropylene glycol moiety, the polypropylene glycol moiety preferablyhas a uniform structure. An exemplary polypropylene glycol moiety havinga uniform structure is as follows:

[0182] This uniform polypropylene glycol structure may be described ashaving only one methyl substituted carbon atom adjacent each oxygen atomin the polypropylene glycol chain. Such uniform polypropylene glycolmoieties may exhibit both lipophilic and hydrophilic characteristics andthus be useful in providing amphiphilic growth hormone drug-oligomerconjugates without the use of lipophilic polymer moieties. Furthermore,coupling the secondary alcohol moiety of the polypropylene glycol moietywith a drug may provide the drug (e.g., a polypeptide) with improvedresistance to degradation caused by enzymes such as trypsin andchymotrypsin found, for example, in the gut.

[0183] Uniform polypropylene glycol according to embodiments of thepresent invention is preferably synthesized as illustrated in FIGS. 11through 13, which will now be described. As illustrated in FIG. 11,1,2-propanediol 53 is reacted with a primary alcohol blocking reagent toprovide a secondary alcohol extension monomer 54. The primary alcoholblocking reagent may be various primary alcohol blocking reagents aswill be understood by those skilled in the art including, but notlimited to, silylchloride compounds such as t-butyldiphenylsilylchlorideand t-butyldimethylsilylchloride, and esterification reagents such asAc₂O. Preferably, the primary alcohol blocking reagent is a primaryalcohol blocking reagent that is substantially non-reactive withsecondary alcohols, such as t-butyldiphenylsilylchloride ort-butyldimethylsilylchloride. The secondary alcohol extension monomer(54) may be reacted with methanesulfonyl chloride (MeSO₂Cl) to provide aprimary extension alcohol monomer mesylate 55.

[0184] Alternatively, the secondary alcohol extension monomer 54 may bereacted with a secondary alcohol blocking reagent to provide compound56. The secondary alcohol blocking reagent may be various secondaryalcohol blocking reagents as will be understood by those skilled in theart including, but not limited to, benzyl chloride. The compound 56 maybe reacted with a B₁ de-blocking reagent to remove the blocking moietyB₁ and provide a primary alcohol extension monomer 57. The B₁de-blocking reagent may be selected from various de-blocking reagents aswill be understood by one skilled in the art. When the primary alcoholhas been blocked by forming an ester, the B₁ de-blocking reagent is ade-esterification reagent, such as a base (e.g., potassium carbonate).When the primary alcohol has been blocked using a silylchloride, the B₁de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).The primary alcohol extension monomer 57 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension monomermesylate 58.

[0185] The primary alcohol extension monomer 54 and the secondaryalcohol extension monomer 57 may be capped as follows. The secondaryalcohol extension monomer 54 may be reacted with a capping reagent toprovide a compound 59. The capping reagent may be various cappingreagents as will be understood by those skilled in the art including,but not limited to, alkyl halides such as methyl chloride. The compound59 may be reacted with a B₁ de-blocking agent as described above toprovide a primary alcohol capping monomer 60. The primary alcoholcapping monomer 60 may be reacted with methane sulfonyl chloride toprovide the secondary alcohol capping monomer mesylate 61. The primaryalcohol extension monomer 57 may be reacted with a capping reagent toprovide a compound 62. The capping reagent may be various cappingreagents as described above. The compound 62 may be reacted with a B₂de-blocking reagent to remove the blocking moiety B₂ and provide asecondary alcohol capping monomer 63. The B₂ de-blocking reagent may bevarious de-blocking agents as will be understood by those skilled in theart including, but not limited to, H₂ in the presence of apalladium/activated carbon catalyst. The secondary alcohol cappingmonomer may be reacted with methanesulfonyl chloride to provide aprimary alcohol capping monomer mesylate 64. While the embodimentsillustrated in FIG. 11 show the synthesis of capping monomers, it is tobe understood that similar reactions may be performed to provide cappingpolymers.

[0186] In general, chain extensions may be effected by reacting aprimary alcohol extension mono- or poly-mer such as the primary alcoholextension monomer 57 with a primary alcohol extension mono- or poly-mermesylate such as the primary alcohol extension monomer mesylate 55 toprovide various uniform polypropylene chains or by reacting a secondaryalcohol extension mono- or poly-mer such as the secondary alcoholextension monomer 54with a secondary alcohol extension mono-or poly-mermesylate such as the secondary alcohol extension monomer mesylate 58.

[0187] For example, in FIG. 13, the primary alcohol extension monomermesylate 55 is reacted with the primary alcohol extension monomer 57 toprovide a dimer compound 65. Alternatively, the secondary alcoholextension monomer mesylate 58 may be reacted with the secondary alcoholextension monomer 54 to provide the dimer compound 65. The B₁ blockingmoiety on the dimer compound 65 may be removed using a B₁ de-blockingreagent as described above to provide a primary alcohol extension dimer66. The primary alcohol extension dimer 66 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension dimermesylate 67. Alternatively, the B₂ blocking moiety on the dimer compound65 may be removed using the B₂ de-blocking reagent as described above toprovide a secondary alcohol extension dimer 69. The secondary alcoholextension dimer 69 may be reacted with methane sulfonyl chloride toprovide a primary alcohol extension dimer mesylate 70.

[0188] As will be understood by those skilled in the art, the chainextension process may be repeated to achieve various other chainlengths. For example, as illustrated in FIG. 13, the primary alcoholextension dimer 66 may be reacted with the primary alcohol extensiondimer mesylate 70 to provide a tetramer compound 72. As furtherillustrated in FIG. 13, a generic chain extension reaction schemeinvolves reacting the primary alcohol extension mono- or poly-mer 73with the primary alcohol extension mono- or poly-mer mesylate 74 toprovide the uniform polypropylene polymer 75. The values of m and n mayeach range from 0 to 1000 or more. Preferably, m and n are each from 0to 50. While the embodiments illustrated in FIG. 13 show primary alcoholextension mono- and/or poly-mers being reacted with primary alcoholextension mono- and/or poly-mer mesylates, it is to be understood thatsimilar reactions may be carried out using secondary alcohol extensionmono- and/or poly-mers and secondary alcohol extension mono- and/orpoly-mer mesylates.

[0189] An end of a primary alcohol extension mono- or poly-mer or an endof a primary alcohol extension mono- or poly-mer mesylate may be reactedwith a primary alcohol capping mono- or poly-mer mesylate or a primaryalcohol capping mono- or poly-mer, respectively, to provide a cappeduniform polypropylene chain. For example, as illustrated in FIG. 12, theprimary alcohol extension dimer mesylate 70 is reacted with the primaryalcohol capping monomer 60 to provide the capped/blocked primary alcoholextension trimer 71. As will be understood by those skilled in the art,the B₁ blocking moiety may be removed and the resulting capped primaryalcohol extension trimer may be reacted with a primary alcohol extensionmono- or poly-mer mesylate to extend the chain of the capped trimer 71.

[0190] An end of a secondary alcohol extension mono-or poly-mer or anend of a secondary alcohol extension mono-or poly-mer mesylate may bereacted with a secondary alcohol capping mono-or poly-mer mesylate or asecondary alcohol capping mono- or poly-mer, respectively, to provide acapped uniform polypropylene chain. For example, as illustrated in FIG.12, the secondary alcohol extension dimer mesylate 67 is reacted withthe secondary alcohol capping monomer 63 to provide the capped/blockedprimary alcohol extension trimer 68. The B₂ blocking moiety may beremoved as described above and the resulting capped secondary alcoholextension trimer may be reacted with a secondary alcohol extension mermesylate to extend the chain of the capped trimer 68. While thesyntheses illustrated in FIG. 12 show the reaction of a dimer with acapping monomer to provide a trimer, it is to be understood that thecapping process may be performed at any point in the synthesis of auniform polypropylene glycol moiety, or, alternatively, uniformpolypropylene glycol moieties may be provided that are not capped. Whilethe embodiments illustrated in FIG. 12 show the capping of apolybutylene oligomer by synthesis with a capping monomer, it is to beunderstood that polybutylene oligomers of the present invention may becapped directly (i.e., without the addition of a capping monomer) usinga capping reagent as described above in FIG. 11.

[0191] Uniform polypropylene glycol moieties according to embodiments ofthe present invention may be coupled to a drug, a lipophilic moiety suchas a carboxylic acid, and/or various other moieties by various methodsas will be understood by those skilled in the art including, but notlimited to, those described herein with respect to polyethylene glycolmoieties.

[0192] The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,hydrophilic moieties, lipophilic moieties, spacer moieties, linkermoieties, and terminating moieties. The various moieties in the oligomerare covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

[0193] The oligomer may further comprise one or more hydrophilicmoieties including, but not limited to, sugars, polyalkylene glycols,and polyamine/PEG copolymers. Adjacent polyalkylene glycol moieties willbe considered to be the same moiety if they are coupled by an ether bondand have the same alkyl structure. For example, the moiety

—O—C₂H₄—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—

[0194] is a single polyethylene glycol moiety having six polyethyleneglycol subunits. Adjacent polyalkylene glycol moieties will beconsidered to be different moieties if they are coupled by a bond otherthan an ether bond or if they have different alkyl structures. Forexample, the moiety

[0195] is a polyethylene glycol moiety having four polyethylene glycolsubunits and a hydrophilic moiety having two polyethylene glycolsubunits. Preferably, oligomers according to embodiments of the presentinvention comprise a polyalkylene glycol moiety and do not furthercomprise a hydrophilic moiety.

[0196] The oligomer may further comprise one or more lipophilic moietiesas will be understood by those skilled in the art. The lipophilic moietyis preferably a saturated or unsaturated, linear or branched alkylmoiety or a saturated or unsaturated, linear or branched fatty acidmoiety. When the lipophilic moiety is an alkyl moiety, it is preferablya linear, saturated or unsaturated alkyl moiety having 1 to 28 carbonatoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. Whenthe lipophilic moiety is a fatty acid moiety, it is preferably a naturalfatty acid moiety that is linear, saturated or unsaturated, having 2 to18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5or 6 carbon atoms.

[0197] The oligomer may further comprise one or more spacer moieties aswill be understood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from thedrug, to separate a first hydrophilic or lipophilic moiety from a secondhydrophilic or lipophilic moiety, or to separate a hydrophilic moiety orlipophilic moiety from a linker moiety. Spacer moieties are preferablyselected from the group consisting of sugar, cholesterol and glycerinemoieties.

[0198] The oligomer may further comprise one or more linker moietiesthat are used to couple the oligomer with the drug as will be understoodby those skilled in the art. Linker moieties are preferably selectedfrom the group consisting of alkyl and fatty acid moieties.

[0199] The oligomer may further comprise one or more terminatingmoieties at the one or more ends of the oligomer which are not coupledto the drug. The terminating moiety is preferably an alkyl or alkoxymoiety, and is more preferably a lower alkyl or lower alkoxy moiety.Most preferably, the terminating moiety is methyl or methoxy. While theterminating moiety is preferably an alkyl or alkoxy moiety, it is to beunderstood that the terminating moiety may be various moieties as willbe understood by those skilled in the art including, but not limited to,sugars, cholesterol, alcohols, and fatty acids.

[0200] The oligomer is preferably covalently coupled to the drug. Insome embodiments, the drug is coupled to the oligomer utilizing ahydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzablecoupling may provide a drug-oligomer conjugate that acts as a prodrug.In certain instances, for example where the drug-oligomer conjugate isinactive (i.e., the conjugate lacks the ability to affect the bodythrough the drug's primary mechanism of action), a hydrolyzable couplingmay provide for a time-release or controlled-release effect,administering the drug over a given time period as one or more oligomersare cleaved from their respective drug-oligomer conjugates to providethe active drug. In other embodiments, the drug is coupled to theoligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, orether bond). Use of a non-hydrolyzable bond may be preferable when it isdesirable to allow the drug-oligomer conjugate to circulate in thebloodstream for an extended period of time, preferably at least 2 hours.

[0201] While the oligomer is preferably covalently coupled to the drug,it is to be understood that the oligomer may be non-covalently coupledto the drug to form a non-covalently conjugated drug-oligomer complex.As will be understood by those skilled in the art, non-covalentcouplings include, but are not limited to, hydrogen bonding, ionicbonding, Van der Waals bonding, and micellular or liposomalencapsulation. According to embodiments of the present invention,oligomers may be suitably constructed, modified and/or appropriatelyfunctionalized to impart the ability for non-covalent conjugation in aselected manner (e.g., to impart hydrogen bonding capability), as willbe understood by those skilled in the art. According to otherembodiments of present invention, oligomers may be derivatized withvarious compounds including, but not limited to, amino acids,oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids,fatty acid derivatives, salicylic acids, salicylic acid derivatives,aminosalicylic acids, and aminosalicylic acid derivatives. The resultingoligomers can non-covalently couple (complex) with drug molecules,pharmaceutical products, and/or pharmaceutical excipients. The resultingcomplexes preferably have balanced lipophilic and hydrophilicproperties. According to still other embodiments of the presentinvention, oligomers may be derivatized with amine and/or alkyl amines.Under suitable acidic conditions, the resulting oligomers can formnon-covalently conjugated complexes with drug molecules, pharmaceuticalproducts and/or pharmaceutical excipients. The products resulting fromsuch complexation preferably have balanced lipophilic and hydrophilicproperties.

[0202] More than one oligomer (i.e., a plurality of oligomers) may becoupled to the drug. The oligomers in the plurality are preferably thesame. However, it is to be understood that the oligomers in theplurality may be different from one another, or, alternatively, some ofthe oligomers in the plurality may be the same and some may bedifferent. When a plurality of oligomers are coupled to the drug, it maybe preferable to couple one or more of the oligomers to the drug withhydrolyzable bonds and couple one or more of the oligomers to the drugwith non-hydrolyzable bonds. Alternatively, all of the bonds couplingthe plurality of oligomers to the drug may be hydrolyzable, but havevarying degrees of hydrolyzability such that, for example, one or moreof the oligomers is rapidly removed from the drug by hydrolysis in thebody and one or more of the oligomers is slowly removed from the drug byhydrolysis in the body.

[0203] The oligomer may be coupled to the drug at various nucleophilicresidues of the drug including, but not limited to, nucleophilichydroxyl functions and/or amino functions. When the drug is apolypeptide, a nucleophilic hydroxyl function may be found, for example,at serine and/or tyrosine residues, and a nucleophilic amino functionmay be found, for example, at histidine and/or lysine residues, and/orat the one or more N-termini of the polypeptide. When an oligomer iscoupled to the one or more N-termini of the polypeptide, the couplingpreferably forms a secondary amine. For example, when the drug is humaninsulin, the oligomer may be coupled to an amino functionality of theinsulin including the amino functionality of Gly^(A1), the aminofunctionality of Phe^(B1), and the amino functionality of Lys^(B29).When one oligomer is coupled to the human insulin, the oligomer ispreferably coupled to the amino functionality of Lys^(B29). When twooligomers are coupled to the human insulin, the oligomers are preferablycoupled to the amino functionality of Phe^(B1) and the aminofunctionality of Lys^(B29). While more than one oligomer may be coupledto the human insulin, a higher activity (improved glucose loweringability) is observed for the mono-conjugated human insulin. As anotherexample, when the drug is salmon calcitonin, the oligomer may be coupledto an amino functionality of the salmon calcitonin, including the aminofunctionality of Lys¹¹, Lys¹⁸ and the N-terminus. While one or moreoligomers may be coupled to the salmon calcitonin, a higher activity(improved glucose lowering ability) is observed for the di-conjugatedsalmon calcitonin where an oligomer is coupled to the aminofunctionality of Lys¹¹ and an oligomer is coupled to the aminofunctionality of Lys¹⁸. As yet another example, when the drug is humangrowth hormone, the oligomer may be coupled to an amino functionality ofPhe¹, Lys³⁸, Lys⁴¹, Lys⁷⁰, Lys¹¹⁵, Lys¹⁴⁰, Lys¹⁴⁵, Lys¹⁵⁸, Lys¹⁶⁸,and/or Lys¹⁷².

[0204] Substantially monodispersed mixtures of drug-oligomer conjugatesof the present invention may be synthesized by various methods. Forexample, a substantially monodispersed mixture of oligomers consistingof carboxylic acid and polyethylene glycol is synthesized by contactinga substantially monodispersed mixture of carboxylic acid with asubstantially monodispersed mixture of polyethylene glycol underconditions sufficient to provide a substantially monodispersed mixtureof oligomers. The oligomers of the substantially monodispersed mixtureare then activated so that they are capable of reacting with a drug toprovide a drug-oligomer conjugate. One embodiment of a synthesis routefor providing a substantially monodispersed mixture of activatedoligomers is illustrated in FIG. 3 and described in Examples 11-18hereinbelow. Another embodiment of a synthesis route for providing asubstantially monodispersed mixture of activated oligomers isillustrated in FIG. 4 and described in Examples 19-24 hereinbelow. Stillanother embodiment of a synthesis route for providing a substantiallymonodispersed mixture of activated oligomers is illustrated in FIG. 5and described in Examples 25-29 hereinbelow. Yet another embodiment of asynthesis route for providing a substantially monodispersed mixture ofactivated oligomers is illustrated in FIG. 6 and described in Examples30-31 hereinbelow. Another embodiment of a synthesis route for providinga substantially monodispersed mixture of activated oligomers isillustrated in FIG. 7 and described in Examples 32-37 hereinbelow. Stillanother embodiment of a synthesis route for providing a substantiallymonodispersed mixture of activated oligomers is illustrated in FIG. 8and described in Example 38 hereinbelow. Yet another embodiment of asynthesis route for providing a substantially monodispersed mixture ofactivated oligomers is illustrated in FIG. 9 and described in Example 39hereinbelow. Another embodiment of a synthesis route for providing asubstantially monodispersed mixture of activated oligomers isillustrated in FIG. 10 and described in Example 40 hereinbelow.

[0205] The substantially monodispersed mixture of activated oligomersmay be reacted with a substantially monodispersed mixture of drugs underconditions sufficient to provide a mixture of drug-oligomer conjugates,as described, for example, in Examples 41-120 hereinbelow. As will beunderstood by those skilled in the art, the reaction conditions (e.g.,selected molar ratios, solvent mixtures and/or pH) may be controlledsuch that the mixture of drug-oligomer conjugates resulting from thereaction of the substantially monodispersed mixture of activatedoligomers and the substantially monodispersed mixture of drugs is asubstantially monodispersed mixture. For example, conjugation at theamino functionality of lysine may be suppressed by maintaining the pH ofthe reaction solution below the pK_(a) of lysine. Alternatively, themixture of drug-oligomer conjugates may be separated and isolatedutilizing, for example, HPLC to provide a substantially monodispersedmixture of drug-oligomer conjugates, for example mono-, di-, ortri-conjugates. The degree of conjugation (e.g., whether the isolatedmolecule is a mono-, di-, or tri-conjugate) of a particular isolatedconjugate may be determined and/or verified utilizing various techniquesas will be understood by those skilled in the art including, but notlimited to, mass spectroscopy. The particular conjugate structure (e.g.,whether the oligomer is at Gly^(A1), Phe^(B1), or Lys^(B29) of a humaninsulin monoconjugate) may be determined and/or verified utilizingvarious techniques as will be understood by those skilled in the artincluding, but not limited to, sequence analysis, peptide mapping,selective enzymatic cleavage, and/or endopeptidase cleavage.

[0206] As will be understood by those skilled in the art, one or more ofthe reaction sites on the drug may be blocked by, for example, reactingthe drug with a suitable blocking reagent such as N-tert-butoxycarbonyl(t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC). This process may bepreferred, for example, when the drug is a polypeptide and it is desiredto form an unsaturated conjugate (i.e., a conjugate wherein not allnucleophilic residues are conjugated) having an oligomer at one or moreof the N-termini of the polypeptide. Following such blocking, thesubstantially monodispersed mixture of blocked drugs may be reacted withthe substantially monodispersed mixture of activated oligomers toprovide a mixture of drug-oligomer conjugates having oligomer(s) coupledto one or more nucleophilic residues and having blocking moietiescoupled to other nucleophilic residues. After the conjugation reaction,the drug-oligomer conjugates may be de-blocked as will be understood bythose skilled in the art. If necessary, the mixture of drug-oligomerconjugates may then be separated as described above to provide asubstantially monodispersed mixture of drug-oligomer conjugates.Alternatively, the mixture of drug-oligomer conjugates may be separatedprior to de-blocking.

[0207] Substantially monodispersed mixtures of drug-oligomer conjugatesaccording to embodiments of the present invention preferably haveimproved properties when compared with those of conventional mixtures.For example, a substantially monodispersed mixture of drug-oligomerconjugates preferably has an in vivo activity that is greater than thein vivo activity of a polydispersed mixture of drug-oligomer conjugateshaving the same number average molecular weight as the substantiallymonodispersed mixture. As will be understood by those skilled in theart, the number average molecular weight of the substantiallymonodispersed mixture and the number average weight of the polydispersedmixture may be measured by various methods including, but not limitedto, size exclusion chromatography such as gel permeation chromatographyas described, for example, in H. R. Allcock & F. W. Lampe, CONTEMPORARYPOLYMER CHEMISTRY 394-402 (2d. ed., 1991).

[0208] As another example, a substantially monodispersed mixture ofdrug-oligomer conjugates preferably has an in vitro activity that isgreater than the in vitro activity of a polydispersed mixture ofdrug-oligomer conjugates having the same number average molecular weightas the substantially monodispersed mixture. As will be understood bythose skilled in the art, the number average molecular weight of thesubstantially monodispersed mixture and the number average weight of thepolydispersed mixture may be measured by various methods including, butnot limited to, size exclusion chromatography such as gel permeationchromatography. The in vitro activity of a particular mixture may bemeasured by various methods, as will be understood by those skilled inthe art. Preferably, the in vitro activity is measured using aCytosensor® Microphysiometer commercially available from MolecularDevices Corporation of Sunnyvale, Calif. The microphysiometer monitorssmall changes in the rates of extracellular acidification in response toa drug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study.

[0209] As still another example, a substantially monodispersed mixtureof drug-oligomer conjugates preferably has an increased resistance todegradation by chymotrypsin when compared to the resistance todegradation by chymotrypsin of a polydispersed mixture of drug-oligomerconjugates having the same number average molecular weight as thesubstantially monodispersed mixture. As will be understood by thoseskilled in the art, the number average molecular weight of thesubstantially monodispersed mixture and the number average weight of thepolydispersed mixture may be measured by various methods including, butnot limited to, size exclusion chromatography.

[0210] As yet another example, a substantially monodispersed mixture ofdrug-oligomer conjugates preferably has an inter-subject variabilitythat is less than the inter-subject variability of a polydispersedmixture of drug-oligomer conjugates having the same number averagemolecular weight as the substantially monodispersed mixture. As will beunderstood by those skilled in the art, the number average molecularweight of the substantially monodispersed mixture and the number averageweight of the polydispersed mixture may be measured by various methodsincluding, but not limited to, size exclusion chromatography. Theinter-subject variability may be measured by various methods as will beunderstood by those skilled in the art. The inter-subject variability ispreferably calculated as follows. The area under a dose response curve(AUC) (i.e., the area between the dose-response curve and a baselinevalue) is determined for each subject. The average AUC for all subjectsis determined by summing the AUCs of each subject and dividing the sumby the number of subjects. The absolute value of the difference betweenthe subject's AUC and the average AUC is then determined for eachsubject. The absolute values of the differences obtained are then summedto give a value that represents the inter-subject variability. Lowervalues represent lower inter-subject variabilities and higher valuesrepresent higher inter-subject variabilities.

[0211] Substantially monodispersed mixtures of drug-oligomer conjugatesaccording to embodiments of the present invention preferably have two ormore of the above-described improved properties. More preferably,substantially monodispersed mixtures of drug-oligomer conjugatesaccording to embodiments of the present invention have three or more ofthe above-described improved properties. Most preferably, substantiallymonodispersed mixtures of drug-oligomer conjugates according toembodiments of the present invention have all four of theabove-described improved properties.

[0212] In still other embodiments according to the present invention, amixture of conjugates having a molecular weight distribution with astandard deviation of less than about 22 Daltons is provided. Eachconjugate in the mixture includes a drug coupled to an oligomer thatcomprises a polyalkylene glycol moiety. The standard deviation ispreferably less than about 14 Daltons and is more preferably less thanabout 11 Daltons. The molecular weight distribution may be determined bymethods known to those skilled in the art including, but not limited to,size exclusion chromatography such as gel permeation chromatography asdescribed, for example, in H. R. Allcock & F. W. Lampe, CONTEMPORARYPOLYMER CHEMISTRY 394-402 (2d. ed., 1991). The standard deviation of themolecular weight distribution may then be determined by statisticalmethods as will be understood by those skilled in the art.

[0213] The oligomer may be various oligomers comprising a polyalkyleneglycol moiety as will be understood by those skilled in the art.Preferably, the polyalkylene glycol moiety has at least 2, 3, or 4polyalkylene glycol subunits. More preferably, the polyalkylene glycolmoiety has at least 5 or 6 polyalkylene glycol subunits. Mostpreferably, the polyalkylene glycol moiety of the oligomer has at least7 polyalkylene glycol subunits. The polyalkylene glycol moiety of theoligomer is preferably a lower alkyl polyalkylene glycol moiety such asa polyethylene glycol moiety, a polypropylene glycol moiety, or apolybutylene glycol moiety. When the polyalkylene moiety is apolypropylene glycol moiety, the polypropylene glycol moiety preferablyhas a uniform structure. An exemplary polypropylene glycol moiety havinga uniform structure is as follows:

[0214] This uniform polypropylene glycol structure may be described ashaving only one methyl substituted carbon atom adjacent each oxygen atomin the polypropylene glycol chain. Such uniform polypropylene glycolmoieties may exhibit both lipophilic and hydrophilic characteristics andthus be useful in providing amphiphilic drug-oligomer conjugates withoutthe use of lipophilic polymer moieties. Furthermore, coupling thesecondary alcohol moiety of the polypropylene glycol moiety with a drugmay provide the drug (e.g., a polypeptide) with improved resistance todegradation caused by enzymes such as trypsin and chymotrypsin found,for example, in the gut.

[0215] Uniform polypropylene glycol according to embodiments of thepresent invention is preferably synthesized as illustrated in FIGS. 11through 13, which will now be described. As illustrated in FIG. 11,1,2-propanediol 53 is reacted with a primary alcohol blocking reagent toprovide a secondary alcohol extension monomer 54. The primary alcoholblocking reagent may be various primary alcohol blocking reagents aswill be understood by those skilled in the art including, but notlimited to, silylchloride compounds such as t-butyldiphenylsilylchlorideand t-butyldimethylsilylchloride, and esterification reagents such asAc₂O. Preferably, the primary alcohol blocking reagent is a primaryalcohol blocking reagent that is substantially non-reactive withsecondary alcohols, such as t-butyldiphenylsilylchloride ort-butyldimethylsilylchloride. The secondary alcohol extension monomer(54) may be reacted with methanesulfonyl chloride (MeSO₂Cl) to provide aprimary extension alcohol monomer mesylate 55.

[0216] Alternatively, the secondary alcohol extension monomer 54 may bereacted with a secondary alcohol blocking reagent to provide compound56. The secondary alcohol blocking reagent may be various secondaryalcohol blocking reagents as will be understood by those skilled in theart including, but not limited to, benzyl chloride. The compound 56 maybe reacted with a B₁ de-blocking reagent to remove the blocking moietyB₁ and provide a primary alcohol extension monomer 57. The B₁de-blocking reagent may be selected from various de-blocking reagents aswill be understood by one skilled in the art. When the primary alcoholhas been blocked by forming an ester, the B₁ de-blocking reagent is ade-esterification reagent, such as a base (e.g., potassium carbonate).When the primary alcohol has been blocked using a silylchloride, the B₁de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).The primary alcohol extension monomer 57 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension monomermesylate 58.

[0217] The primary alcohol extension monomer 54 and the secondaryalcohol extension monomer 57 may be capped as follows. The secondaryalcohol extension monomer 54 may be reacted with a capping reagent toprovide a compound 59. The capping reagent may be various cappingreagents as will be understood by those skilled in the art including,but not limited to, alkyl halides such as methyl chloride. The compound59 may be reacted with a B₁ de-blocking agent as described above toprovide a primary alcohol capping monomer 60. The primary alcoholcapping monomer 60 may be reacted with methane sulfonyl chloride toprovide the secondary alcohol capping monomer mesylate 61. The primaryalcohol extension monomer 57 may be reacted with a capping reagent toprovide a compound 62. The capping reagent may be various cappingreagents as described above. The compound 62 may be reacted with a B₂de-blocking reagent to remove the blocking moiety B₂ and provide asecondary alcohol capping monomer 63. The B₂ de-blocking reagent may bevarious de-blocking agents as will be understood by those skilled in theart including, but not limited to, H₂ in the presence of apalladium/activated carbon catalyst. The secondary alcohol cappingmonomer may be reacted with methanesulfonyl chloride to provide aprimary alcohol capping monomer mesylate 64. While the embodimentsillustrated in FIG. 11 show the synthesis of capping monomers, it is tobe understood that similar reactions may be performed to provide cappingpolymers.

[0218] In general, chain extensions may be effected by reacting aprimary alcohol extension mono- or poly-mer such as the primary alcoholextension monomer 57 with a primary alcohol extension mono- or poly-mermesylate such as the primary alcohol extension monomer mesylate 55 toprovide various uniform polypropylene chains or by reacting a secondaryalcohol extension mono- or poly-mer such as the secondary alcoholextension monomer 54 with a secondary alcohol extension mono-or poly-mermesylate such as the secondary alcohol extension monomer mesylate 58.

[0219] For example, in FIG. 13, the primary alcohol extension monomermesylate 55 is reacted with the primary alcohol extension monomer 57 toprovide a dimer compound 65. Alternatively, the secondary alcoholextension monomer mesylate 58 may be reacted with the secondary alcoholextension monomer 54 to provide the dimer compound 65. The B₁ blockingmoiety on the dimer compound 65 may be removed using a B₁ de-blockingreagent as described above to provide a primary alcohol extension dimer66. The primary alcohol extension dimer 66 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension dimermesylate 67. Alternatively, the B₂ blocking moiety on the dimer compound65 may be removed using the B₂ de-blocking reagent as described above toprovide a secondary alcohol extension dimer 69. The secondary alcoholextension dimer 69 may be reacted with methane sulfonyl chloride toprovide a primary alcohol extension dimer mesylate 70.

[0220] As will be understood by those skilled in the art, the chainextension process may be repeated to achieve various other chainlengths. For example, as illustrated in FIG. 13, the primary alcoholextension dimer 66 may be reacted with the primary alcohol extensiondimer mesylate 70 to provide a tetramer compound 72. As furtherillustrated in FIG. 13, a generic chain extension reaction schemeinvolves reacting the primary alcohol extension mono- or poly-mer 73with the primary alcohol extension mono- or poly-mer mesylate 74 toprovide the uniform polypropylene polymer 75. The values of m and n mayeach range from 0 to 1000 or more. Preferably, m and n are each from 0to 50. While the embodiments illustrated in FIG. 13 show primary alcoholextension mono- and/or poly-mers being reacted with primary alcoholextension mono- and/or poly-mer mesylates, it is to be understood thatsimilar reactions may be carried out using secondary alcohol extensionmono- and/or poly-mers and secondary alcohol extension mono- and/orpoly-mer mesylates.

[0221] An end of a primary alcohol extension mono- or poly-mer or an endof a primary alcohol extension mono- or poly-mer mesylate may be reactedwith a primary alcohol capping mono- or poly-mer mesylate or a primaryalcohol capping mono- or poly-mer, respectively, to provide a cappeduniform polypropylene chain. For example, as illustrated in FIG. 12, theprimary alcohol extension dimer mesylate 70 is reacted with the primaryalcohol capping monomer 60 to provide the capped/blocked primary alcoholextension trimer 71. As will be understood by those skilled in the art,the B₁ blocking moiety may be removed and the resulting capped primaryalcohol extension trimer may be reacted with a primary alcohol extensionmono- or poly-mer mesylate to extend the chain of the capped trimer 71.

[0222] An end of a secondary alcohol extension mono-or poly-mer or anend of a secondary alcohol extension mono-or poly-mer mesylate may bereacted with a secondary alcohol capping mono-or poly-mer mesylate or asecondary alcohol capping mono- or poly-mer, respectively, to provide acapped uniform polypropylene chain. For example, as illustrated in FIG.12, the secondary alcohol extension dimer mesylate 67 is reacted withthe secondary alcohol capping monomer 63 to provide the capped/blockedprimary alcohol extension trimer 68. The B₂ blocking moiety may beremoved as described above and the resulting capped secondary alcoholextension trimer may be reacted with a secondary alcohol extension mermesylate to extend the chain of the capped trimer 68. While thesyntheses illustrated in FIG. 12 show the reaction of a dimer with acapping monomer to provide a trimer, it is to be understood that thecapping process may be performed at any point in the synthesis of auniform polypropylene glycol moiety, or, alternatively, uniformpolypropylene glycol moieties may be provided that are not capped. Whilethe embodiments illustrated in FIG. 12 show the capping of apolybutylene oligomer by synthesis with a capping monomer, it is to beunderstood that polybutylene oligomers of the present invention may becapped directly (i.e., without the addition of a capping monomer) usinga capping reagent as described above in FIG. 11.

[0223] Uniform polypropylene glycol moieties according to embodiments ofthe present invention may be coupled to a drug, a lipophilic moiety suchas a carboxylic acid, and/or various other moieties by various methodsas will be understood by those skilled in the art including, but notlimited to, those described herein with respect to polyethylene glycolmoieties.

[0224] The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,hydrophilic moieties, lipophilic moieties, spacer moieties, linkermoieties, and terminating moieties. The various moieties in the oligomerare covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

[0225] The oligomer may further comprise one or more hydrophilicmoieties including, but not limited to, sugars, polyalkylene glycols,and polyamine/PEG copolymers. Adjacent polyalkylene glycol moieties willbe considered to be the same moiety if they are coupled by an ether bondand have the same alkyl structure. For example, the moiety

—O—C₂H₄—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—

[0226] is a single polyethylene glycol moiety having six polyethyleneglycol subunits. Adjacent polyalkylene glycol moieties will beconsidered to be different moieties if they are coupled by a bond otherthan an ether bond or if they have different alkyl structures. Forexample, the moiety

[0227] is a polyethylene glycol moiety having four polyethylene glycolsubunits and a hydrophilic moiety having two polyethylene glycolsubunits. Preferably, oligomers according to embodiments of the presentinvention comprise a polyalkylene glycol moiety and do not furthercomprise a hydrophilic moiety.

[0228] The oligomer may further comprise one or more lipophilic moietiesas will be understood by those skilled in the art. The lipophilic moietyis preferably a saturated or unsaturated, linear or branched alkylmoiety or a saturated or unsaturated, linear or branched fatty acidmoiety. When the lipophilic moiety is an alkyl moiety, it is preferablya linear, saturated or unsaturated alkyl moiety having 1 to 28 carbonatoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. Whenthe lipophilic moiety is a fatty acid moiety, it is preferably a naturalfatty acid moiety that is linear, saturated or unsaturated, having 2 to18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5or 6 carbon atoms.

[0229] The oligomer may further comprise one or more spacer moieties aswill be understood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from thedrug, to separate a first hydrophilic or lipophilic moiety from a secondhydrophilic or lipophilic moiety, or to separate a hydrophilic moiety orlipophilic moiety from a linker moiety. Spacer moieties are preferablyselected from the group consisting of sugar, cholesterol and glycerinemoieties.

[0230] The oligomer may further comprise one or more linker moietiesthat are used to couple the oligomer with the drug as will be understoodby those skilled in the art. Linker moieties are preferably selectedfrom the group consisting of alkyl and fatty acid moieties.

[0231] The oligomer may further comprise one or more terminatingmoieties at the one or more ends of the oligomer which are not coupledto the drug. The terminating moiety is preferably an alkyl or alkoxymoiety, and is more preferably a lower alkyl or lower alkoxy moiety.Most preferably, the terminating moiety is methyl or methoxy. While theterminating moiety is preferably an alkyl or alkoxy moiety, it is to beunderstood that the terminating moiety may be various moieties as willbe understood by those skilled in the art including, but not limited to,sugars, cholesterol, alcohols, and fatty acids.

[0232] The oligomer is preferably covalently coupled to the drug. Insome embodiments, the drug is coupled to the oligomer utilizing ahydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzablecoupling may provide a drug-oligomer conjugate that acts as a prodrug.In certain instances, for example where the drug-oligomer conjugate isinactive (i.e., the conjugate lacks the ability to affect the bodythrough the drug's primary mechanism of action), a hydrolyzable couplingmay provide for a time-release or controlled-release effect,administering the drug over a given time period as one or more oligomersare cleaved from their respective drug-oligomer conjugates to providethe active drug. In other embodiments, the drug is coupled to theoligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, orether bond). Use of a non-hydrolyzable bond may be preferable when it isdesirable to allow the drug-oligomer conjugate to circulate in thebloodstream for an extended period of time, preferably at least 2 hours.

[0233] While the oligomer is preferably covalently coupled to the drug,it is to be understood that the oligomer may be non-covalently coupledto the drug to form a non-covalently conjugated drug-oligomer complex.As will be understood by those skilled in the art, non-covalentcouplings include, but are not limited to, hydrogen bonding, ionicbonding, Van der Waals bonding, and micellular or liposomalencapsulation. According to embodiments of the present invention,oligomers may be suitably constructed, modified and/or appropriatelyfinctionalized to impart the ability for non-covalent conjugation in aselected manner (e.g., to impart hydrogen bonding capability), as willbe understood by those skilled in the art. According to otherembodiments of present invention, oligomers may be derivatized withvarious compounds including, but not limited to, amino acids,oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids,fatty acid derivatives, salicylic acids, salicylic acid derivatives,aminosalicylic acids, and aminosalicylic acid derivatives. The resultingoligomers can non-covalently couple (complex) with drug molecules,pharmaceutical products, and/or pharmaceutical excipients. The resultingcomplexes preferably have balanced lipophilic and hydrophilicproperties. According to still other embodiments of the presentinvention, oligomers may be derivatized with amine and/or alkyl amines.Under suitable acidic conditions, the resulting oligomers can formnon-covalently conjugated complexes with drug molecules, pharmaceuticalproducts and/or pharmaceutical excipients. The products resulting fromsuch complexation preferably have balanced lipophilic and hydrophilicproperties.

[0234] More than one oligomer (i.e., a plurality of oligomers) may becoupled to the drug. The oligomers in the plurality are preferably thesame. However, it is to be understood that the oligomers in theplurality may be different from one another, or, alternatively, some ofthe oligomers in the plurality may be the same and some may bedifferent. When a plurality of oligomers are coupled to the drug, it maybe preferable to couple one or more of the oligomers to the drug withhydrolyzable bonds and couple one or more of the oligomers to the drugwith non-hydrolyzable bonds. Alternatively, all of the bonds couplingthe plurality of oligomers to the drug may be hydrolyzable, but havevarying degrees of hydrolyzability such that, for example, one or moreof the oligomers is rapidly removed from the drug by hydrolysis in thebody and one or more of the oligomers is slowly removed from the drug byhydrolysis in the body.

[0235] The oligomer may be coupled to the drug at various nucleophilicresidues of the drug including, but not limited to, nucleophilichydroxyl functions and/or amino functions. When the drug is apolypeptide, a nucleophilic hydroxyl function may be found, for example,at serine and/or tyrosine residues, and a nucleophilic amino functionmay be found, for example, at histidine and/or lysine residues, and/orat the one or more N-termini of the polypeptide. When an oligomer iscoupled to the one or more N-termini of the polypeptide, the couplingpreferably forms a secondary amine. For example, when the drug is humaninsulin, the oligomer may be coupled to an amino functionality of theinsulin including the amino functionality of Gly^(A1), the aminofunctionality of Phe^(B1), and the amino functionality of Lys^(B29).When one oligomer is coupled to the human insulin, the oligomer ispreferably coupled to the amino functionality of LyS^(B29). When twooligomers are coupled to the human insulin, the oligomers are preferablycoupled to the amino functionality of Phe^(B1) and the aminofunctionality of LyS^(B29). While more than one oligomer may be coupledto the human insulin, a higher activity (improved glucose loweringability) is observed for the mono-conjugated human insulin. As anotherexample, when the drug is salmon calcitonin, the oligomer may be coupledto an amino functionality of the salmon calcitonin, including the aminofunctionality of Lys¹¹, Lys¹⁸ and the N-terminus. While one or moreoligomers may be coupled to the salmon calcitonin, a higher activity(improved glucose lowering ability) is observed for the di-conjugatedsalmon calcitonin where an oligomer is coupled to the aminofunctionality of Lys¹¹ and an oligomer is coupled to the aminofunctionality of Lys¹⁸. As yet another example, when the drug is humangrowth hormone, the oligomer may be coupled to an amino functionality ofPhe¹, Lys³⁸, Lys⁴¹, Lys⁷⁰, Lys¹¹⁵, Lys¹⁴⁰, Lys¹⁴⁵, Lys¹⁵⁸, Lys¹⁶⁸,and/or Lys¹⁷².

[0236] Mixtures of drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltons maybe synthesized by various methods. For example, a mixture of oligomershaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons consisting of carboxylic acid and polyethyleneglycol is synthesized by contacting a mixture of carboxylic acid havinga molecular weight distribution with a standard deviation of less thanabout 22 Daltons with a mixture of polyethylene glycol having amolecular weight distribution with a standard deviation of less thanabout 22 Daltons under conditions sufficient to provide a mixture ofoligomers having a molecular weight distribution with a standarddeviation of less than about 22 Daltons. The oligomers of the mixturehaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons are then activated so that they are capable ofreacting with a drug to provide a drug-oligomer conjugate. Oneembodiment of a synthesis route for providing a mixture of activatedoligomers having a molecular weight distribution with a standarddeviation of less than about 22 Daltons is illustrated in FIG. 3 anddescribed in Examples 11-18 hereinbelow. Another embodiment of asynthesis route for providing a mixture of activated oligomers having amolecular weight distribution with a standard deviation of less thanabout 22 Daltons is illustrated in FIG. 4 and described in Examples19-24 hereinbelow. Still another embodiment of a synthesis route forproviding a mixture of activated oligomers having a molecular weightdistribution with a standard deviation of less than about 22 Daltons isillustrated in FIG. 5 and described in Examples 25-29 hereinbelow. Yetanother embodiment of a synthesis route for providing a mixture ofactivated oligomers having a molecular weight distribution with astandard deviation of less than about 22 Daltons is illustrated in FIG.6 and described in Examples 30-31 hereinbelow. Another embodiment of asynthesis route for providing a mixture of activated oligomers having amolecular weight distribution with a standard deviation of less thanabout 22 Daltons is illustrated in FIG. 7 and described in Examples32-37 hereinbelow. Still another embodiment of a synthesis route forproviding a mixture of activated oligomers having a molecular weightdistribution with a standard deviation of less than about 22 Daltons isillustrated in FIG. 8 and described in Example 38 hereinbelow. Yetanother embodiment of a synthesis route for providing a mixture ofactivated oligomers having a molecular weight distribution with astandard deviation of less than about 22 Daltons is illustrated in FIG.9 and described in Example 39 hereinbelow. Another embodiment of asynthesis route for providing a mixture of activated oligomers having amolecular weight distribution with a standard deviation of less thanabout 22 Daltons is illustrated in FIG. 10 and described in Example 40hereinbelow.

[0237] The mixture of activated oligomers having a molecular weightdistribution with a standard deviation of less than about 22 Daltons maybe reacted with a mixture of drugs having a standard deviation of lessthan about 22 Daltons under conditions sufficient to provide a mixtureof drug-oligomer conjugates, as described, for example, in Examples41-120 hereinbelow. As will be understood by those skilled in the art,the reaction conditions (e.g., selected molar ratios, solvent mixturesand/or pH) may be controlled such that the mixture of drug-oligomerconjugates resulting from the reaction of the mixture of activatedoligomers having a molecular weight distribution with a standarddeviation of less than about 22 Daltons and the mixture of drugs havinga molecular weight distribution with a standard deviation of less thanabout 22 Daltons is a mixture having a molecular weight distributionwith a standard deviation of less than about 22 Daltons. For example,conjugation at the amino functionality of lysine may be suppressed bymaintaining the pH of the reaction solution below the pK_(a) of lysine.Alternatively, the mixture of drug-oligomer conjugates may be separatedand isolated utilizing, for example, HPLC to provide a mixture ofdrug-oligomer conjugates, for example mono-, di-, or tri-conjugates,having a molecular weight distribution with a standard deviation of lessthan about 22 Daltons. The degree of conjugation (e.g., whether theisolated molecule is a mono-, di-, or tri-conjugate) of a particularisolated conjugate may be determined and/or verified utilizing varioustechniques as will be understood by those skilled in the art including,but not limited to, mass spectroscopy. The particular conjugatestructure (e.g., whether the oligomer is at Gly^(A1), Phe^(B1), orLys^(B29) of a human insulin monoconjugate) may be determined and/orverified utilizing various techniques as will be understood by thoseskilled in the art including, but not limited to, sequence analysis,peptide mapping, selective enzymatic cleavage, and/or endopeptidasecleavage.

[0238] As will be understood by those skilled in the art, one or more ofthe reaction sites on the drug may be blocked by, for example, reactingthe drug with a suitable blocking reagent such as N-tert-butoxycarbonyl(t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC). This process may bepreferred, for example, when the drug is a polypeptide and it is desiredto form an unsaturated conjugate (i.e., a conjugate wherein not allnucleophilic residues are conjugated) having one or more oligomers atthe one or more N-termini of the polypeptide. Following such blocking,the mixture of blocked drugs having a molecular weight distribution witha standard deviation of less than about 22 Daltons may be reacted withthe mixture of activated oligomers having a molecular weightdistribution with a standard deviation of less than about 22 Daltons toprovide a mixture of drug-oligomer conjugates having oligomer(s) coupledto one or more nucleophilic residues and having blocking moietiescoupled to other nucleophilic residues. After the conjugation reaction,the drug-oligomer conjugates may be de-blocked as will be understood bythose skilled in the art. If necessary, the mixture of drug-oligomerconjugates may then be separated as described above to provide a mixtureof drug-oligomer conjugates having a molecular weight distribution witha standard deviation of less than about 22 Daltons. Alternatively, themixture of drug-oligomer conjugates may be separated prior tode-blocking.

[0239] Mixtures of drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltonsaccording to embodiments of the present invention preferably haveimproved properties when compared with those of conventional mixtures.For example, a mixture of drug-oligomer conjugates having a molecularweight distribution with a standard deviation of less than about 22Daltons preferably has an in vivo activity that is greater than the invivo activity of a polydispersed mixture of drug-oligomer conjugateshaving the same number average molecular weight as the mixture ofdrug-oligomer conjugates having a molecular weight distribution with astandard deviation of less than about 22 Daltons. As will be understoodby those skilled in the art, the number average molecular weight of themixture of drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltons andthe number average weight of the polydispersed mixture may be measuredby various methods including, but not limited to, size exclusionchromatography such as gel permeation chromatography as described, forexample, in H. R. Allcock & F. W. Lampe, CONTEMPORARY POLYMER CHEMISTRY394-402 (2d. ed., 1991).

[0240] As another example, a mixture of drug-oligomer conjugates havinga molecular weight distribution with a standard deviation of less thanabout 22 Daltons preferably has an in vitro activity that is greaterthan the in vitro activity of a polydispersed mixture of drug-oligomerconjugates having the same number average molecular weight as themixture of drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltons. Aswill be understood by those skilled in the art, the number averagemolecular weight of the mixture of drug-oligomer conjugates having amolecular weight distribution with a standard deviation of less thanabout 22 Daltons and the number average weight of the polydispersedmixture may be measured by various methods including, but not limitedto, size exclusion chromatography such as gel permeation chromatography.

[0241] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study.

[0242] As still another example, a mixture of drug-oligomer conjugateshaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons preferably has an increased resistance todegradation by chymotrypsin when compared to the resistance todegradation by chymotrypsin of a polydispersed mixture of drug-oligomerconjugates having the same number average molecular weight as themixture of drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltons. Aswill be understood by those skilled in the art, the number averagemolecular weight of the mixture of drug-oligomer conjugates having amolecular weight distribution with a standard deviation of less thanabout 22 Daltons and the number average weight of the polydispersedmixture may be measured by various methods including, but not limitedto, size exclusion chromatography.

[0243] As yet another example, a mixture of drug-oligomer conjugateshaving a molecular weight distribution with a standard deviation of lessthan about 22 Daltons preferably has an inter-subject variability thatis less than the inter-subject variability of a polydispersed mixture ofdrug-oligomer conjugates having the same number average molecular weightas the mixture of drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltons. Aswill be understood by those skilled in the art, the number averagemolecular weight of the mixture of drug-oligomer conjugates having amolecular weight distribution with a standard deviation of less thanabout 22 Daltons and the number average weight of the polydispersedmixture may be measured by various methods including, but not limitedto, size exclusion chromatography. The inter-subject variability may bemeasured by various methods as will be understood by those skilled inthe art. The inter-subject variability is preferably calculated asfollows. The area under a dose response curve (AUC) (i.e., the areabetween the dose-response curve and a baseline value) is determined foreach subject. The average AUC for all subjects is determined by summingthe AUCs of each subject and dividing the sum by the number of subjects.The absolute value of the difference between the subject's AUC and theaverage AUC is then determined for each subject. The absolute values ofthe differences obtained are then summed to give a value that representsthe inter-subject variability. Lower values represent lowerinter-subject variabilities and higher values represent higherinter-subject variabilities.

[0244] Mixtures of drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltonsaccording to embodiments of the present invention preferably have two ormore of the above-described improved properties. More preferably,mixtures of drug-oligomer conjugates having a molecular weightdistribution with a standard deviation of less than about 22 Daltonsaccording to embodiments of the present invention have three or more ofthe above-described improved properties. Most preferably, mixtures ofdrug-oligomer conjugates having a molecular weight distribution with astandard deviation of less than about 22 Daltons according toembodiments of the present invention have all four of theabove-described improved properties.

[0245] According to yet other embodiments of the present invention, amixture of conjugates is provided where each conjugate includes a drugcoupled to an oligomer that comprises a polyalkylene glycol moiety, andthe mixture has a dispersity coefficient (DC) greater than 10,000 where${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}\quad N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}}} \right)^{2}}$

[0246] wherein:

[0247] n is the number of different molecules in the sample;

[0248] N_(i) is the number of i^(th) molecules in the sample; and

[0249] M_(i) is the mass of the i^(th) molecule.

[0250] The mixture of conjugates preferably has a dispersity coefficientgreater than 100,000. More preferably, the dispersity coefficient of theconjugate mixture is greater than 500,000 and, most preferably, thedispersity coefficient is greater than 10,000,000. The variables n,N_(i), and M_(i) may be determined by various methods as will beunderstood by those skilled in the art, including, but not limited to,methods described below in Example 123.

[0251] The oligomer may be various oligomers comprising a polyalkyleneglycol moiety as will be understood by those skilled in the art.Preferably, the polyalkylene glycol moiety has at least 2, 3, or 4polyalkylene glycol subunits. More preferably, the polyalkylene glycolmoiety has at least 5 or 6 polyalkylene glycol subunits. Mostpreferably, the polyalkylene glycol moiety of the oligomer has at least7 polyalkylene glycol subunits. The polyalkylene glycol moiety of theoligomer is preferably a lower alkyl polyalkylene glycol moiety such asa polyethylene glycol moiety, a polypropylene glycol moiety, or apolybutylene glycol moiety. When the polyalkylene moiety is apolypropylene glycol moiety, the polypropylene glycol moiety preferablyhas a uniform structure. An exemplary polypropylene glycol moiety havinga uniform structure is as follows:

[0252] This uniform polypropylene glycol structure may be described ashaving only one methyl substituted carbon atom adjacent each oxygen atomin the polypropylene glycol chain. Such uniform polypropylene glycolmoieties may exhibit both lipophilic and hydrophilic characteristics andthus be useful in providing amphiphilic drug-oligomer conjugates withoutthe use of lipophilic polymer moieties. Furthermore, coupling thesecondary alcohol moiety of the polypropylene glycol moiety with a drugmay provide the drug (e.g., a polypeptide) with improved resistance todegradation caused by enzymes such as trypsin and chymotrypsin found,for example, in the gut.

[0253] Uniform polypropylene glycol according to embodiments of thepresent invention is preferably synthesized as illustrated in FIGS. 11through 13, which will now be described. As illustrated in FIG. 11,1,2-propanediol 53 is reacted with a primary alcohol blocking reagent toprovide a secondary alcohol extension monomer 54. The primary alcoholblocking reagent may be various primary alcohol blocking reagents aswill be understood by those skilled in the art including, but notlimited to, silylchloride compounds such as t-butyldiphenylsilylchlorideand t-butyldimethylsilylchloride, and esterification reagents such asAc₂O. Preferably, the primary alcohol blocking reagent is a primaryalcohol blocking reagent that is substantially non-reactive withsecondary alcohols, such as t-butyldiphenylsilylchloride ort-butyldimethylsilylchloride. The secondary alcohol extension monomer(54) may be reacted with methanesulfonyl chloride (MeSO₂Cl) to provide aprimary extension alcohol monomer mesylate 55.

[0254] Alternatively, the secondary alcohol extension monomer 54 may bereacted with a secondary alcohol blocking reagent to provide compound56. The secondary alcohol blocking reagent may be various secondaryalcohol blocking reagents as will be understood by those skilled in theart including, but not limited to, benzyl chloride. The compound 56 maybe reacted with a B₁ de-blocking reagent to remove the blocking moietyB₁ and provide a primary alcohol extension monomer 57. The B₁de-blocking reagent may be selected from various de-blocking reagents aswill be understood by one skilled in the art. When the primary alcoholhas been blocked by forming an ester, the B₁ de-blocking reagent is ade-esterification reagent, such as a base (e.g., potassium carbonate).When the primary alcohol has been blocked using a silylchloride, the B₁de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).The primary alcohol extension monomer 57 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension monomermesylate 58.

[0255] The primary alcohol extension monomer 54 and the secondaryalcohol extension monomer 57 may be capped as follows. The secondaryalcohol extension monomer 54 may be reacted with a capping reagent toprovide a compound 59. The capping reagent may be various cappingreagents as will be understood by those skilled in the art including,but not limited to, alkyl halides such as methyl chloride. The compound59 may be reacted with a B₁ de-blocking agent as described above toprovide a primary alcohol capping monomer 60. The primary alcoholcapping monomer 60 may be reacted with methane sulfonyl chloride toprovide the secondary alcohol capping monomer mesylate 61. The primaryalcohol extension monomer 57 may be reacted with a capping reagent toprovide a compound 62. The capping reagent may be various cappingreagents as described above. The compound 62 may be reacted with a B₂de-blocking reagent to remove the blocking moiety B₂ and provide asecondary alcohol capping monomer 63. The B₂ de-blocking reagent may bevarious de-blocking agents as will be understood by those skilled in theart including, but not limited to, H₂ in the presence of apalladium/activated carbon catalyst. The secondary alcohol cappingmonomer may be reacted with methanesulfonyl chloride to provide aprimary alcohol capping monomer mesylate 64. While the embodimentsillustrated in FIG. 11 show the synthesis of capping monomers, it is tobe understood that similar reactions may be performed to provide cappingpolymers.

[0256] In general, chain extensions may be effected by reacting aprimary alcohol extension mono- or poly-mer such as the primary alcoholextension monomer 57 with a primary alcohol extension mono- or poly-mermesylate such as the primary alcohol extension monomer mesylate 55 toprovide various uniform polypropylene chains or by reacting a secondaryalcohol extension mono- or poly-mer such as the secondary alcoholextension monomer 54 with a secondary alcohol extension mono-or poly-mermesylate such as the secondary alcohol extension monomer mesylate 58.

[0257] For example, in FIG. 13, the primary alcohol extension monomermesylate 55 is reacted with the primary alcohol extension monomer 57 toprovide a dimer compound 65. Alternatively, the secondary alcoholextension monomer mesylate 58 may be reacted with the secondary alcoholextension monomer 54 to provide the dimer compound 65. The B₁ blockingmoiety on the dimer compound 65 may be removed using a B₁ de-blockingreagent as described above to provide a primary alcohol extension dimer66. The primary alcohol extension dimer 66 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension dimermesylate 67. Alternatively, the B₂ blocking moiety on the dimer compound65 may be removed using the B₂ de-blocking reagent as described above toprovide a secondary alcohol extension dimer 69. The secondary alcoholextension dimer 69 may be reacted with methane sulfonyl chloride toprovide a primary alcohol extension dimer mesylate 70.

[0258] As will be understood by those skilled in the art, the chainextension process may be repeated to achieve various other chainlengths. For example, as illustrated in FIG. 13, the primary alcoholextension dimer 66 may be reacted with the primary alcohol extensiondimer mesylate 70 to provide a tetramer compound 72. As furtherillustrated in FIG. 13, a generic chain extension reaction schemeinvolves reacting the primary alcohol extension mono- or poly-mer 73with the primary alcohol extension mono- or poly-mer mesylate 74 toprovide the uniform polypropylene polymer 75. The values of m and n mayeach range from 0 to 1000 or more. Preferably, m and n are each from 0to 50. While the embodiments illustrated in FIG. 13 show primary alcoholextension mono- and/or poly-mers being reacted with primary alcoholextension mono- and/or poly-mer mesylates, it is to be understood thatsimilar reactions may be carried out using secondary alcohol extensionmono- and/or poly-mers and secondary alcohol extension mono- and/orpoly-mer mesylates.

[0259] An end of a primary alcohol extension mono- or poly-mer or an endof a primary alcohol extension mono- or poly-mer mesylate may be reactedwith a primary alcohol capping mono- or poly-mer mesylate or a primaryalcohol capping mono- or poly-mer, respectively, to provide a cappeduniform polypropylene chain. For example, as illustrated in FIG. 12, theprimary alcohol extension dimer mesylate 70 is reacted with the primaryalcohol capping monomer 60 to provide the capped/blocked primary alcoholextension trimer 71. As will be understood by those skilled in the art,the B₁ blocking moiety may be removed and the resulting capped primaryalcohol extension trimer may be reacted with a primary alcohol extensionmono- or poly-mer mesylate to extend the chain of the capped trimer 71.

[0260] An end of a secondary alcohol extension mono-or poly-mer or anend of a secondary alcohol extension mono-or poly-mer mesylate may bereacted with a secondary alcohol capping mono-or poly-mer mesylate or asecondary alcohol capping mono- or poly-mer, respectively, to provide acapped uniform polypropylene chain. For example, as illustrated in FIG.12, the secondary alcohol extension dimer mesylate 67 is reacted withthe secondary alcohol capping monomer 63 to provide the capped/blockedprimary alcohol extension trimer 68. The B₂ blocking moiety may beremoved as described above and the resulting capped secondary alcoholextension trimer may be reacted with a secondary alcohol extension mermesylate to extend the chain of the capped trimer 68. While thesyntheses illustrated in FIG. 12 show the reaction of a dimer with acapping monomer to provide a trimer, it is to be understood that thecapping process may be performed at any point in the synthesis of auniform polypropylene glycol moiety, or, alternatively, uniformpolypropylene glycol moieties may be provided that are not capped. Whilethe embodiments illustrated in FIG. 12 show the capping of apolybutylene oligomer by synthesis with a capping monomer, it is to beunderstood that polybutylene oligomers of the present invention may becapped directly (i.e., without the addition of a capping monomer) usinga capping reagent as described above in FIG. 11.

[0261] Uniform polypropylene glycol moieties according to embodiments ofthe present invention may be coupled to a drug, a lipophilic moiety suchas a carboxylic acid, and/or various other moieties by various methodsas will be understood by those skilled in the art including, but notlimited to, those described herein with respect to polyethylene glycolmoieties.

[0262] The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,hydrophilic moieties, lipophilic moieties, spacer moieties, linkermoieties, and terminating moieties. The various moieties in the oligomerare covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

[0263] The oligomer may further comprise one or more hydrophilicmoieties including, but not limited to, sugars, polyalkylene glycols,and polyamine/PEG copolymers. Adjacent polyalkylene glycol moieties willbe considered to be the same moiety if they are coupled by an ether bondand have the same alkyl structure. For example, the moiety

—O—C₂H₄—O—C₂H₄—O—C₂H₄—C₂H₄—O—C₂H₄—O—C₂H₄—

[0264] is a single polyethylene glycol moiety having six polyethyleneglycol subunits. Adjacent polyalkylene glycol moieties will beconsidered to be different moieties if they are coupled by a bond otherthan an ether bond or if they have different alkyl structures. Forexample, the moiety

[0265] is a polyethylene glycol moiety having four polyethylene glycolsubunits and a hydrophilic moiety having two polyethylene glycolsubunits. Preferably, oligomers according to embodiments of the presentinvention comprise a polyalkylene glycol moiety and do not furthercomprise a hydrophilic moiety.

[0266] The oligomer may further comprise one or more lipophilic moietiesas will be understood by those skilled in the art. The lipophilic moietyis preferably a saturated or unsaturated, linear or branched alkylmoiety or a saturated or unsaturated, linear or branched fatty acidmoiety. When the lipophilic moiety is an alkyl moiety, it is preferablya linear, saturated or unsaturated alkyl moiety having 1 to 28 carbonatoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. Whenthe lipophilic moiety is a fatty acid moiety, it is preferably a naturalfatty acid moiety that is linear, saturated or unsaturated, having 2 to18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5or 6 carbon atoms.

[0267] The oligomer may further comprise one or more spacer moieties aswill be understood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from thedrug, to separate a first hydrophilic or lipophilic moiety from a secondhydrophilic or lipophilic moiety, or to separate a hydrophilic moiety orlipophilic moiety from a linker moiety. Spacer moieties are preferablyselected from the group consisting of sugar, cholesterol and glycerinemoieties.

[0268] The oligomer may further comprise one or more linker moietiesthat are used to couple the oligomer with the drug as will be understoodby those skilled in the art. Linker moieties are preferably selectedfrom the group consisting of alkyl and fatty acid moieties.

[0269] The oligomer may further comprise one or more terminatingmoieties at the one or more ends of the oligomer which are not coupledto the drug. The terminating moiety is preferably an alkyl or alkoxymoiety, and is more preferably a lower alkyl or lower alkoxy moiety.Most preferably, the terminating moiety is methyl or methoxy. While theterminating moiety is preferably an alkyl or alkoxy moiety, it is to beunderstood that the terminating moiety may be various moieties as willbe understood by those skilled in the art including, but not limited to,sugars, cholesterol, alcohols, and fatty acids.

[0270] The oligomer is preferably covalently coupled to the drug. Insome embodiments, the drug is coupled to the oligomer utilizing ahydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzablecoupling may provide a drug-oligomer conjugate that acts as a prodrug.In certain instances, for example where the drug-oligomer conjugate isinactive (i.e., the conjugate lacks the ability to affect the bodythrough the drug's primary mechanism of action), a hydrolyzable couplingmay provide for a time-release or controlled-release effect,administering the drug over a given time period as one or more oligomersare cleaved from their respective drug-oligomer conjugates to providethe active drug. In other embodiments, the drug is coupled to theoligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, orether bond). Use of a non-hydrolyzable bond may be preferable when it isdesirable to allow the drug-oligomer conjugate to circulate in thebloodstream for an extended period of time, preferably at least 2 hours.

[0271] While the oligomer is preferably covalently coupled to the drug,it is to be understood that the oligomer may be non-covalently coupledto the drug to form a non-covalently conjugated drug-oligomer complex.As will be understood by those skilled in the art, non-covalentcouplings include, but are not limited to, hydrogen bonding, ionicbonding, Van der Waals bonding, and micellular or liposomalencapsulation. According to embodiments of the present invention,oligomers may be suitably constructed, modified and/or appropriatelyfunctionalized to impart the ability for non-covalent conjugation in aselected manner (e.g., to impart hydrogen bonding capability), as willbe understood by those skilled in the art. According to otherembodiments of present invention, oligomers may be derivatized withvarious compounds including, but not limited to, amino acids,oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids,fatty acid derivatives, salicylic acids, salicylic acid derivatives,aminosalicylic acids, and aminosalicylic acid derivatives. The resultingoligomers can non-covalently couple (complex) with drug molecules,pharmaceutical products, and/or pharmaceutical excipients. The resultingcomplexes preferably have balanced lipophilic and hydrophilicproperties. According to still other embodiments of the presentinvention, oligomers may be derivatized with amine and/or alkyl amines.Under suitable acidic conditions, the resulting oligomers can formnon-covalently conjugated complexes with drug molecules, pharmaceuticalproducts and/or pharmaceutical excipients. The products resulting fromsuch complexation preferably have balanced lipophilic and hydrophilicproperties.

[0272] More than one oligomer (i.e., a plurality of oligomers) may becoupled to the drug. The oligomers in the plurality are preferably thesame. However, it is to be understood that the oligomers in theplurality may be different from one another, or, alternatively, some ofthe oligomers in the plurality may be the same and some may bedifferent. When a plurality of oligomers are coupled to the drug, it maybe preferable to couple one or more of the oligomers to the drug withhydrolyzable bonds and couple one or more of the oligomers to the drugwith non-hydrolyzable bonds. Alternatively, all of the bonds couplingthe plurality of oligomers to the drug may be hydrolyzable, but havevarying degrees of hydrolyzability such that, for example, one or moreof the oligomers is rapidly removed from the drug by hydrolysis in thebody and one or more of the oligomers is slowly removed from the drug byhydrolysis in the body.

[0273] The oligomer may be coupled to the drug at various nucleophilicresidues of the drug including, but not limited to, nucleophilichydroxyl functions and/or amino functions. When the drug is apolypeptide, a nucleophilic hydroxyl function may be found, for example,at serine and/or tyrosine residues, and a nucleophilic amino functionmay be found, for example, at histidine and/or lysine residues, and/orat the one or more N-termini of the polypeptide. When an oligomer iscoupled to the one or more N-termini of the polypeptide, the couplingpreferably forms a secondary amine. For example, when the drug is humaninsulin, the oligomer may be coupled to an amino functionality of theinsulin including the amino functionality of Gly^(A1), the aminofunctionality of Phe^(B1), and the amino functionality of Lys^(B29).When one oligomer is coupled to the human insulin, the oligomer ispreferably coupled to the amino functionality of Lys^(B29). When twooligomers are coupled to the human insulin, the oligomers are preferablycoupled to the amino functionality of Phe^(B1) and the aminofunctionality of Lys^(B29). While more than one oligomer may be coupledto the human insulin, a higher activity (improved glucose loweringability) is observed for the mono-conjugated human insulin. As anotherexample, when the drug is salmon calcitonin, the oligomer may be coupledto an amino functionality of the salmon calcitonin, including the aminofunctionality of Lys¹¹, Lys¹⁸ and the N-terminus. While one or moreoligomers may be coupled to the salmon calcitonin, a higher activity(improved glucose lowering ability) is observed for the di-conjugatedsalmon calcitonin where an oligomer is coupled to the aminofunctionality of Lys¹¹ and an oligomer is coupled to the aminofunctionality of Lys¹⁸. As yet another example, when the drug is humangrowth hormone, the oligomer may be coupled to an amino functionality ofPhe¹, Lys³⁸, Lys⁴¹, Lys⁷⁰, Lys¹¹⁵, Lys¹⁴⁰, Lys¹⁴⁵, Lys¹⁵⁸, Lys¹⁶⁸,and/or Lys¹⁷².

[0274] Mixtures of drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 may be synthesized by various methods.For example, a mixture of oligomers having a dispersity coefficientgreater than 10,000 consisting of carboxylic acid and polyethyleneglycol is synthesized by contacting a mixture of carboxylic acid havinga dispersity coefficient greater than 10,000 with a mixture ofpolyethylene glycol having a dispersity coefficient greater than 10,000under conditions sufficient to provide a mixture of oligomers having adispersity coefficient greater than 10,000. The oligomers of the mixturehaving a dispersity coefficient greater than 10,000 are then activatedso that they are capable of reacting with a drug to provide adrug-oligomer conjugate. One embodiment of a synthesis route forproviding a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 3 and describedin Examples 11-18 hereinbelow. Another embodiment of a synthesis routefor providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 4 and describedin Examples 19-24 hereinbelow. Still another embodiment of a synthesisroute for providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 5 and describedin Examples 25-29 hereinbelow. Yet another embodiment of a synthesisroute for providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 6 and describedin Examples 30-31 hereinbelow. Another embodiment of a synthesis routefor providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 7 and describedin Examples 32-37 hereinbelow. Still another embodiment of a synthesisroute for providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 8 and describedin Example 38 hereinbelow. Yet another embodiment of a synthesis routefor providing a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 9 and describedin Example 39 hereinbelow. Another embodiment of a synthesis route forproviding a mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is illustrated in FIG. 10 and describedin Example 40 hereinbelow.

[0275] The mixture of activated oligomers having a dispersitycoefficient greater than 10,000 is reacted with a mixture of drugshaving a dispersity coefficient greater than 10,000 under conditionssufficient to provide a mixture of drug-oligomer conjugates, asdescribed, for example, in Examples 41-120 hereinbelow. As will beunderstood by those skilled in the art, the reaction conditions (e.g.,selected molar ratios, solvent mixtures and/or pH) may be controlledsuch that the mixture of drug-oligomer conjugates resulting from thereaction of the mixture of activated oligomers having a dispersitycoefficient greater than 10,000 and the mixture of drugs having adispersity coefficient greater than 10,000 is a mixture having adispersity coefficient greater than 10,000. For example, conjugation atthe amino functionality of lysine may be suppressed by maintaining thepH of the reaction solution below the pK_(a) of lysine. Alternatively,the mixture of drug-oligomer conjugates may be separated and isolatedutilizing, for example, HPLC to provide a mixture of drug-oligomerconjugates, for example mono-, di-, or tri-conjugates, having adispersity coefficient greater than 10,000. The degree of conjugation(e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate)of a particular isolated conjugate may be determined and/or verifiedutilizing various techniques as will be understood by those skilled inthe art including, but not limited to, mass spectroscopy. The particularconjugate structure (e.g., whether the oligomer is at Gly^(A1)Phe^(B1),or Lys^(B29) of a human insulin monoconjugate) may be determined and/orverified utilizing various techniques as will be understood by thoseskilled in the art including, but not limited to, sequence analysis,peptide mapping, selective enzymatic cleavage, and/or endopeptidasecleavage.

[0276] As will be understood by those skilled in the art, one or more ofthe reaction sites on the drug may be blocked by, for example, reactingthe drug with a suitable blocking reagent such as N-tert-butoxycarbonyl(t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC). This process may bepreferred, for example, when the drug is a polypeptide and it is desiredto form an unsaturated conjugate (i.e., a conjugate wherein not allnucleophilic residues are conjugated) having an oligomer at the one ormore N-termini of the polypeptide. Following such blocking, the mixtureof blocked drugs having a dispersity coefficient greater than 10,000 maybe reacted with the mixture of activated oligomers having a dispersitycoefficient greater than 10,000 to provide a mixture of drug-oligomerconjugates having oligomer(s) coupled to one or more nucleophilicresidues and having blocking moieties coupled to other nucleophilicresidues. After the conjugation reaction, the drug-oligomer conjugatesmay be de-blocked as will be understood by those skilled in the art. Ifnecessary, the mixture of drug-oligomer conjugates may then be separatedas described above to provide a mixture of drug-oligomer conjugateshaving a dispersity coefficient greater than 10,000. Alternatively, themixture of drug-oligomer conjugates may be separated prior tode-blocking.

[0277] Mixtures of drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 according to embodiments of the presentinvention preferably have improved properties when compared with thoseof conventional mixtures. For example, a mixture of drug-oligomerconjugates having a dispersity coefficient greater than 10,000preferably has an in vivo activity that is greater than the in vivoactivity of a polydispersed mixture of drug-oligomer conjugates havingthe same number average molecular weight as the mixture of drug-oligomerconjugates having a dispersity coefficient greater than 10,000. As willbe understood by those skilled in the art, the number average molecularweight of the mixture of drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 and the number average molecular weightof the polydispersed mixture may be measured by various methodsincluding, but not limited to, size exclusion chromatography such as gelpermeation chromatography as described, for example, in H. R. Allcock &F. W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).

[0278] As another example, a mixture of drug-oligomer conjugates havinga dispersity coefficient greater than 10,000 preferably has an in vitroactivity that is greater than the in vitro activity of a polydispersedmixture of drug-oligomer conjugates having the same number averagemolecular weight as the mixture of drug-oligomer conjugates having adispersity coefficient greater than 10,000. As will be understood bythose skilled in the art, the number average molecular weight of themixture of drug-oligomer conjugates having a dispersity coefficientgreater than 10,000 and the number average weight of the polydispersedmixture may be measured by various methods including, but not limitedto, size exclusion chromatography.

[0279] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study.

[0280] As still another example, a mixture of drug-oligomer conjugateshaving a dispersity coefficient greater than 10,000 preferably has anincreased resistance to degradation by chymotrypsin when compared to theresistance to degradation by chymotrypsin of a polydispersed mixture ofdrug-oligomer conjugates having the same number average molecular weightas the mixture of drug-oligomer conjugates having a dispersitycoefficient greater than 10,000. As will be understood by those skilledin the art, the number average molecular weight of the mixture ofdrug-oligomer conjugates having a dispersity coefficient greater than10,000 and the number average weight of the polydispersed mixture may bemeasured by various methods including, but not limited to, sizeexclusion chromatography.

[0281] As yet another example, a mixture of drug-oligomer conjugateshaving a dispersity coefficient greater than 10,000 preferably has aninter-subject variability that is less than the inter-subjectvariability of a polydispersed mixture of drug-oligomer conjugateshaving the same number average molecular weight as the mixture ofdrug-oligomer conjugates having a dispersity coefficient greater than10,000. As will be understood by those skilled in the art, the numberaverage molecular weight of the mixture of drug-oligomer conjugateshaving a dispersity coefficient greater than 10,000 and the numberaverage weight of the polydispersed mixture may be measured by variousmethods including, but not limited to, size exclusion chromatography.The inter-subject variability may be measured by various methods as willbe understood by those skilled in the art. The inter-subject variabilityis preferably calculated as follows. The area under a dose responsecurve (AUC) (i.e., the area between the dose-response curve and abaseline value) is determined for each subject. The average AUC for allsubjects is determined by summing the AUCs of each subject and dividingthe sum by the number of subjects. The absolute value of the differencebetween the subject's AUC and the average AUC is then determined foreach subject. The absolute values of the differences obtained are thensummed to give a value that represents the inter-subject variability.Lower values represent lower inter-subject variabilities and highervalues represent higher inter-subject variabilities.

[0282] Mixtures of drug-oligomer conjugates having a dispersitycoefficient greater than 10,000 according to embodiments of the presentinvention preferably have two or more of the above-described improvedproperties. More preferably, mixtures of drug-oligomer conjugates havinga dispersity coefficient greater than 10,000 according to embodiments ofthe present invention have three or more of the above-described improvedproperties. Most preferably, mixtures of drug-oligomer conjugates havinga dispersity coefficient greater than 10,000 according to embodiments ofthe present invention have all four of the above-described improvedproperties.

[0283] According to other embodiments of the present invention, amixture of conjugates in which each conjugate includes a drug coupled toan oligomer and has the same number of polyalkylene glycol subunits isprovided.

[0284] The oligomer may be various oligomers comprising a polyalkyleneglycol moiety as will be understood by those skilled in the art.Preferably, the polyalkylene glycol moiety has at least 2, 3, or 4polyalkylene glycol subunits. More preferably, the polyalkylene glycolmoiety has at least 5 or 6 polyalkylene glycol subunits. Mostpreferably, the polyalkylene glycol moiety of the oligomer has at least7 polyalkylene glycol subunits. The polyalkylene glycol moiety of theoligomer is preferably a lower alkyl polyalkylene glycol moiety such asa polyethylene glycol moiety, a polypropylene glycol moiety, or apolybutylene glycol moiety. When the polyalkylene moiety is apolypropylene glycol moiety, the polypropylene glycol moiety preferablyhas a uniform structure. An exemplary polypropylene glycol moiety havinga uniform structure is as follows:

[0285] This uniform polypropylene glycol structure may be described ashaving only one methyl substituted carbon atom adjacent each oxygen atomin the polypropylene glycol chain. Such uniform polypropylene glycolmoieties may exhibit both lipophilic and hydrophilic characteristics andthus be useful in providing amphiphilic drug-oligomer conjugates withoutthe use of lipophilic polymer moieties. Furthermore, coupling thesecondary alcohol moiety of the polypropylene glycol moiety with a drugmay provide the drug (e.g., a polypeptide) with improved resistance todegradation caused by enzymes such as trypsin and chymotrypsin found,for example, in the gut.

[0286] Uniform polypropylene glycol according to embodiments of thepresent invention is preferably synthesized as illustrated in FIGS. 11through 13, which will now be described. As illustrated in FIG. 11,1,2-propanediol 53 is reacted with a primary alcohol blocking reagent toprovide a secondary alcohol extension monomer 54. The primary alcoholblocking reagent may be various primary alcohol blocking reagents aswill be understood by those skilled in the art including, but notlimited to, silylchloride compounds such as t-butyldiphenylsilylchlorideand t-butyldimethylsilylchloride, and esterification reagents such asAc₂O. Preferably, the primary alcohol blocking reagent is a primaryalcohol blocking reagent that is substantially non-reactive withsecondary alcohols, such as t-butyldiphenylsilylchloride ort-butyldimethylsilylchloride. The secondary alcohol extension monomer(54) may be reacted with methanesulfonyl chloride (MeSO₂Cl) to provide aprimary extension alcohol monomer mesylate 55.

[0287] Alternatively, the secondary alcohol extension monomer 54 may bereacted with a secondary alcohol blocking reagent to provide compound56. The secondary alcohol blocking reagent may be various secondaryalcohol blocking reagents as will be understood by those skilled in theart including, but not limited to, benzyl chloride. The compound 56 maybe reacted with a B₁ de-blocking reagent to remove the blocking moietyB₁ and provide a primary alcohol extension monomer 57. The B₁de-blocking reagent may be selected from various de-blocking reagents aswill be understood by one skilled in the art. When the primary alcoholhas been blocked by forming an ester, the B₁ de-blocking reagent is ade-esterification reagent, such as a base (e.g., potassium carbonate).When the primary alcohol has been blocked using a silylchloride, the B₁de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).The primary alcohol extension monomer 57 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension monomermesylate 58.

[0288] The primary alcohol extension monomer 54 and the secondaryalcohol extension monomer 57 may be capped as follows. The secondaryalcohol extension monomer 54 may be reacted with a capping reagent toprovide a compound 59. The capping reagent may be various cappingreagents as will be understood by those skilled in the art including,but not limited to, alkyl halides such as methyl chloride. The compound59 may be reacted with a B₁ de-blocking agent as described above toprovide a primary alcohol capping monomer 60. The primary alcoholcapping monomer 60 may be reacted with methane sulfonyl chloride toprovide the secondary alcohol capping monomer mesylate 61. The primaryalcohol extension monomer 57 may be reacted with a capping reagent toprovide a compound 62. The capping reagent may be various cappingreagents as described above. The compound 62 may be reacted with a B₂de-blocking reagent to remove the blocking moiety B₂ and provide asecondary alcohol capping monomer 63. The B₂ de-blocking reagent may bevarious de-blocking agents as will be understood by those skilled in theart including, but not limited to, H₂ in the presence of apalladium/activated carbon catalyst. The secondary alcohol cappingmonomer may be reacted with methanesulfonyl chloride to provide aprimary alcohol capping monomer mesylate 64. While the embodimentsillustrated in FIG. 11 show the synthesis of capping monomers, it is tobe understood that similar reactions may be performed to provide cappingpolymers.

[0289] In general, chain extensions may be effected by reacting aprimary alcohol extension mono- or poly-mer such as the primary alcoholextension monomer 57 with a primary alcohol extension mono- or poly-mermesylate such as the primary alcohol extension monomer mesylate 55 toprovide various uniform polypropylene chains or by reacting a secondaryalcohol extension mono- or poly-mer such as the secondary alcoholextension monomer 54 with a secondary alcohol extension mono-or poly-mermesylate such as the secondary alcohol extension monomer mesylate 58.

[0290] For example, in FIG. 13, the primary alcohol extension monomermesylate 55 is reacted with the primary alcohol extension monomer 57 toprovide a dimer compound 65. Alternatively, the secondary alcoholextension monomer mesylate 58 may be reacted with the secondary alcoholextension monomer 54 to provide the dimer compound 65. The B₁ blockingmoiety on the dimer compound 65 may be removed using a B₁ de-blockingreagent as described above to provide a primary alcohol extension dimer66. The primary alcohol extension dimer 66 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension dimermesylate 67. Alternatively, the B₂ blocking moiety on the dimer compound65 may be removed using the B₂ de-blocking reagent as described above toprovide a secondary alcohol extension dimer 69. The secondary alcoholextension dimer 69 may be reacted with methane sulfonyl chloride toprovide a primary alcohol extension dimer mesylate 70.

[0291] As will be understood by those skilled in the art, the chainextension process may be repeated to achieve various other chainlengths. For example, as illustrated in FIG. 13, the primary alcoholextension dimer 66 may be reacted with the primary alcohol extensiondimer mesylate 70 to provide a tetramer compound 72. As furtherillustrated in FIG. 13, a generic chain extension reaction schemeinvolves reacting the primary alcohol extension mono- or poly-mer 73with the primary alcohol extension mono- or poly-mer mesylate 74 toprovide the uniform polypropylene polymer 75. The values of m and n mayeach range from 0 to 1000 or more. Preferably, m and n are each from 0to 50. While the embodiments illustrated in FIG. 13 show primary alcoholextension mono- and/or poly-mers being reacted with primary alcoholextension mono- and/or poly-mer mesylates, it is to be understood thatsimilar reactions may be carried out using secondary alcohol extensionmono- and/or poly-mers and secondary alcohol extension mono- and/orpoly-mer mesylates.

[0292] An end of a primary alcohol extension mono- or poly-mer or an endof a primary alcohol extension mono- or poly-mer mesylate may be reactedwith a primary alcohol capping mono- or poly-mer mesylate or a primaryalcohol capping mono- or poly-mer, respectively, to provide a cappeduniform polypropylene chain. For example, as illustrated in FIG. 12, theprimary alcohol extension dimer mesylate 70 is reacted with the primaryalcohol capping monomer 60 to provide the capped/blocked primary alcoholextension trimer 71. As will be understood by those skilled in the art,the B₁ blocking moiety may be removed and the resulting capped primaryalcohol extension trimer may be reacted with a primary alcohol extensionmono- or poly-mer mesylate to extend the chain of the capped trimer 71.

[0293] An end of a secondary alcohol extension mono-or poly-mer or anend of a secondary alcohol extension mono-or poly-mer mesylate may bereacted with a secondary alcohol capping mono-or poly-mer mesylate or asecondary alcohol capping mono- or poly-mer, respectively, to provide acapped uniform polypropylene chain. For example, as illustrated in FIG.12, the secondary alcohol extension dimer mesylate 67 is reacted withthe secondary alcohol capping monomer 63 to provide the capped/blockedprimary alcohol extension trimer 68. The B₂ blocking moiety may beremoved as described above and the resulting capped secondary alcoholextension trimer may be reacted with a secondary alcohol extension mermesylate to extend the chain of the capped trimer 68. While thesyntheses illustrated in FIG. 12 show the reaction of a dimer with acapping monomer to provide a trimer, it is to be understood that thecapping process may be performed at any point in the synthesis of auniform polypropylene glycol moiety, or, alternatively, uniformpolypropylene glycol moieties may be provided that are not capped. Whilethe embodiments illustrated in FIG. 12 show the capping of apolybutylene oligomer by synthesis with a capping monomer, it is to beunderstood that polybutylene oligomers of the present invention may becapped directly (i.e., without the addition of a capping monomer) usinga capping reagent as described above in FIG. 11.

[0294] Uniform polypropylene glycol moieties according to embodiments ofthe present invention may be coupled to a drug, a lipophilic moiety suchas a carboxylic acid, and/or various other moieties by various methodsas will be understood by those skilled in the art including, but notlimited to, those described herein with respect to polyethylene glycolmoieties.

[0295] The oligomer may comprise one or more other moieties as will beunderstood by those skilled in the art including, but not limited to,hydrophilic moieties, lipophilic moieties, spacer moieties, linkermoieties, and terminating moieties. The various moieties in the oligomerare covalently coupled to one another by either hydrolyzable ornon-hydrolyzable bonds.

[0296] The oligomer may further comprise one or more hydrophilicmoieties including, but not limited to, sugars, polyalkylene glycols,and polyamine/PEG copolymers. Adjacent polyalkylene glycol moieties willbe considered to be the same moiety if they are coupled by an ether bondand have the same alkyl structure. For example, the moiety

—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—O—C₂H₄—

[0297] is a single polyethylene glycol moiety having six polyethyleneglycol subunits. Adjacent polyalkylene glycol moieties will beconsidered to be different moieties if they are coupled by a bond otherthan an ether bond or if they have different alkyl structures. Forexample, the moiety

[0298] is a polyethylene glycol moiety having four polyethylene glycolsubunits and a hydrophilic moiety having two polyethylene glycolsubunits. Preferably, oligomers according to embodiments of the presentinvention comprise a polyalkylene glycol moiety and do not furthercomprise a hydrophilic moiety.

[0299] The oligomer may further comprise one or more lipophilic moietiesas will be understood by those skilled in the art. The lipophilic moietyis preferably a saturated or unsaturated, linear or branched alkylmoiety or a saturated or unsaturated, linear or branched fatty acidmoiety. When the lipophilic moiety is an alkyl moiety, it is preferablya linear, saturated or unsaturated alkyl moiety having 1 to 28 carbonatoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms. Whenthe lipophilic moiety is a fatty acid moiety, it is preferably a naturalfatty acid moiety that is linear, saturated or unsaturated, having 2 to18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5or 6 carbon atoms.

[0300] The oligomer may further comprise one or more spacer moieties aswill be understood by those skilled in the art. Spacer moieties may, forexample, be used to separate a hydrophilic moiety from a lipophilicmoiety, to separate a lipophilic moiety or hydrophilic moiety from thedrug, to separate a first hydrophilic or lipophilic moiety from a secondhydrophilic or lipophilic moiety, or to separate a hydrophilic moiety orlipophilic moiety from a linker moiety. Spacer moieties are preferablyselected from the group consisting of sugar, cholesterol and glycerinemoieties.

[0301] The oligomer may further comprise one or more linker moietiesthat are used to couple the oligomer with the drug as will be understoodby those skilled in the art. Linker moieties are preferably selectedfrom the group consisting of alkyl and fatty acid moieties.

[0302] The oligomer may further comprise one or more terminatingmoieties at the one or more ends of the oligomer which are not coupledto the drug. The terminating moiety is preferably an alkyl or alkoxymoiety, and is more preferably a lower alkyl or lower alkoxy moiety.Most preferably, the terminating moiety is methyl or methoxy. While theterminating moiety is preferably an alkyl or alkoxy moiety, it is to beunderstood that the terminating moiety may be various moieties as willbe understood by those skilled in the art including, but not limited to,sugars, cholesterol, alcohols, and fatty acids.

[0303] The oligomer is preferably covalently coupled to the drug. Insome embodiments, the drug is coupled to the oligomer utilizing ahydrolyzable bond (e.g., an ester or carbonate bond). A hydrolyzablecoupling may provide a drug-oligomer conjugate that acts as a prodrug.In certain instances, for example where the drug-oligomer conjugate isinactive (i.e., the conjugate lacks the ability to affect the bodythrough the drug's primary mechanism of action), a hydrolyzable couplingmay provide for a time-release or controlled-release effect,administering the drug over a given time period as one or more oligomersare cleaved from their respective drug-oligomer conjugates to providethe active drug. In other embodiments, the drug is coupled to theoligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, orether bond). Use of a non-hydrolyzable bond may be preferable when it isdesirable to allow the drug-oligomer conjugate to circulate in thebloodstream for an extended period of time, preferably at least 2 hours.

[0304] While the oligomer is preferably covalently coupled to the drug,it is to be understood that the oligomer may be non-covalently coupledto the drug to form a non-covalently conjugated drug-oligomer complex.As will be understood by those skilled in the art, non-covalentcouplings include, but are not limited to, hydrogen bonding, ionicbonding, Van der Waals bonding, and micellular or liposomalencapsulation. According to embodiments of the present invention,oligomers may be suitably constructed, modified and/or appropriatelyfunctionalized to impart the ability for non-covalent conjugation in aselected manner (e.g., to impart hydrogen bonding capability), as willbe understood by those skilled in the art. According to otherembodiments of present invention, oligomers may be derivatized withvarious compounds including, but not limited to, amino acids,oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids,fatty acid derivatives, salicylic acids, salicylic acid derivatives,aminosalicylic acids, and aminosalicylic acid derivatives. The resultingoligomers can non-covalently couple (complex) with drug molecules,pharmaceutical products, and/or pharmaceutical excipients. The resultingcomplexes preferably have balanced lipophilic and hydrophilicproperties. According to still other embodiments of the presentinvention, oligomers may be derivatized with amine and/or alkyl amines.Under suitable acidic conditions, the resulting oligomers can formnon-covalently conjugated complexes with drug molecules, pharmaceuticalproducts and/or pharmaceutical excipients. The products resulting fromsuch complexation preferably have balanced lipophilic and hydrophilicproperties.

[0305] More than one oligomer (i.e., a plurality of oligomers) may becoupled to the drug. The oligomers in the plurality are preferably thesame. However, it is to be understood that the oligomers in theplurality may be different from one another, or, alternatively, some ofthe oligomers in the plurality may be the same and some may bedifferent. When a plurality of oligomers are coupled to the drug, it maybe preferable to couple one or more of the oligomers to the drug withhydrolyzable bonds and couple one or more of the oligomers to the drugwith non-hydrolyzable bonds. Alternatively, all of the bonds couplingthe plurality of oligomers to the drug may be hydrolyzable, but havevarying degrees of hydrolyzability such that, for example, one or moreof the oligomers is rapidly removed from the drug by hydrolysis in thebody and one or more of the oligomers is slowly removed from the drug byhydrolysis in the body.

[0306] The oligomer may be coupled to the drug at various nucleophilicresidues of the drug including, but not limited to, nucleophilichydroxyl finctions and/or amino functions. When the drug is apolypeptide, a nucleophilic hydroxyl fuiction may be found, for example,at serine and/or tyrosine residues, and a nucleophilic amino finctionmay be found, for example, at histidine and/or lysine residues, and/orat the one or more N-termini of the polypeptide. When an oligomer iscoupled to the one or more N-termini of the polypeptide, the couplingpreferably forms a secondary amine. For example, when the drug is humaninsulin, the oligomer may be coupled to an amino functionality of theinsulin including the amino functionality of Gly^(A1), the aminofunctionality of Phe^(B1), and the amino functionality of Lys^(B29).When one oligomer is coupled to the human insulin, the oligomer ispreferably coupled to the amino functionality of Lys^(B29). When twooligomers are coupled to the human insulin, the oligomers are preferablycoupled to the amino functionality of Phe^(B1) and the aminofunctionality of Lys^(B29). While more than one oligomer may be coupledto the human insulin, a higher activity (improved glucose loweringability) is observed for the mono-conjugated human insulin. As anotherexample, when the drug is salmon calcitonin, the oligomer may be coupledto an amino functionality of the salmon calcitonin, including the aminofunctionality of Lys¹¹, Lys¹⁸ and the N-terminus. While one or moreoligomers may be coupled to the salmon calcitonin, a higher activity(improved glucose lowering ability) is observed for the di-conjugatedsalmon calcitonin where an oligomer is coupled to the aminofunctionality of Lys¹¹ and an oligomer is coupled to the aminofunctionality of Lys¹⁸. As yet another example, when the drug is humangrowth hormone, the oligomer may be coupled to an amino functionality ofPhe¹, Lys³⁸, Lys⁴¹, Lys⁷⁰, Lys¹¹⁵, Lys¹⁴⁰, Lys¹⁴⁵, Lys¹⁵⁸, Lys¹⁶⁸,and/or Lys¹⁷².

[0307] Mixtures of drug oligomer conjugates where each conjugate in themixture has the same number of polyethylene glycol subunits may besynthesized by various methods. For example, a mixture of oligomersconsisting of carboxylic acid and polyethylene glycol where eacholigomer in the mixture has the same number of polyethylene glycolsubunits is synthesized by contacting a mixture of carboxylic acid witha mixture of polyethylene glycol where each polyethylene glycol moleculein the mixture has the same number of polyethylene glycol subunits underconditions sufficient to provide a mixture of oligomers where eacholigomer in the mixture has the same number of polyethylene glycolsubunits. The oligomers of the mixture where each oligomer in themixture has the same number of polyethylene glycol subunits are thenactivated so that they are capable of reacting with a drug to provide adrug-oligomer conjugate. One embodiment of a synthesis route forproviding a mixture of activated oligomers where each oligomer in themixture has the same number of polyethylene glycol subunits isillustrated in FIG. 3 and described in Examples 11-18 hereinbelow.Another embodiment of a synthesis route for providing a mixture ofactivated oligomers where each oligomer in the mixture has the samenumber of polyethylene glycol subunits is illustrated in FIG. 4 anddescribed in Examples 19-24 hereinbelow. Still another embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer in the mixture has the same number of polyethylene glycolsubunits is illustrated in FIG. 5 and described in Examples 25-29hereinbelow. Yet another embodiment of a synthesis route for providing amixture of activated oligomers where each oligomer in the mixture hasthe same number of polyethylene glycol subunits is illustrated in FIG. 6and described in Examples 30-31 hereinbelow. Another embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer in the mixture has the same number of polyethylene glycolsubunits is illustrated in FIG. 7 and described in Examples 32-37hereinbelow. Still another embodiment of a synthesis route for providinga mixture of activated oligomers where each oligomer in the mixture hasthe same number of polyethylene glycol subunits is illustrated in FIG. 8and described in Example 38 hereinbelow. Yet another embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer in the mixture has the same number of polyethylene glycolsubunits is illustrated in FIG. 9 and described in Example 39hereinbelow. Another embodiment of a synthesis route for providing amixture of activated oligomers having a mixture of activated oligomerswhere each oligomer in the mixture has the same number of polyethyleneglycol subunits is illustrated in FIG. 10 and described in Example 40hereinbelow.

[0308] The mixture of activated oligomers where each oligomer in themixture has the same number of polyethylene glycol subunits is reactedwith a mixture of drugs under conditions sufficient to provide a mixtureof drug-oligomer conjugates, as described, for example, in Examples41-120 hereinbelow. As will be understood by those skilled in the art,the reaction conditions (e.g., selected molar ratios, solvent mixturesand/or pH) may be controlled such that the mixture of drug-oligomerconjugates resulting from the reaction of the mixture of activatedoligomers where each oligomer in the mixture has the same number ofpolyethylene glycol subunits and the mixture of drugs is a mixture ofconjugates where each conjugate in the mixture has the same number ofpolyethylene glycol subunits. For example, conjugation at the aminofunctionality of lysine may be suppressed by maintaining the pH of thereaction solution below the pK_(a) of lysine. Alternatively, the mixtureof drug-oligomer conjugates may be separated and isolated utilizing, forexample, HPLC to provide a mixture of drug-oligomer conjugates, forexample mono-, di-, or tri-conjugates, where each conjugate in themixture has the same number of polyethylene glycol subunits. The degreeof conjugation (e.g., whether the isolated molecule is a mono-, di-, ortri-conjugate) of a particular isolated conjugate may be determinedand/or verified utilizing various techniques as will be understood bythose skilled in the art including, but not limited to, massspectroscopy. The particular conjugate structure (e.g., whether theoligomer is at Gly^(A1), Phe^(B1), Lys^(B29) of a human insulinmonoconjugate) may be determined and/or verified utilizing varioustechniques as will be understood by those skilled in the art including,but not limited to, sequence analysis, peptide mapping, selectiveenzymatic cleavage, and/or endopeptidase cleavage.

[0309] As will be understood by those skilled in the art, one or more ofthe reaction sites on the drug may be blocked by, for example, reactingthe drug with a suitable blocking reagent such as N-tert-butoxycarbonyl(t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC). This process may bepreferred, for example, when the drug is a polypeptide and it is desiredto form an unsaturated conjugate (i.e., a conjugate wherein not allnucleophilic residues are conjugated) having an oligomer at the one ormore N-termini of the polypeptide. Following such blocking, the mixtureof blocked drugs may be reacted with the mixture of activated oligomerswhere each oligomer in the mixture has the same number of polyethyleneglycol subunits to provide a mixture of drug-oligomer conjugates havingoligomer(s) coupled to one or more nucleophilic residues and havingblocking moieties coupled to other nucleophilic residues. After theconjugation reaction, the drug-oligomer conjugates may be de-blocked aswill be understood by those skilled in the art. If necessary, themixture of drug-oligomer conjugates may then be separated as describedabove to provide a mixture of drug-oligomer conjugates where eachconjugate in the mixture has the same number of polyethylene glycolsubunits. Alternatively, the mixture of drug-oligomer conjugates may beseparated prior to de-blocking.

[0310] Mixtures of drug-oligomer conjugates where each conjugate in themixture has the same number of polyethylene glycol subunits according toembodiments of the present invention preferably have improved propertieswhen compared with those of conventional mixtures. For example, amixture of drug-oligomer conjugates where each conjugate in the mixturehas the same number of polyethylene glycol subunits preferably has an invivo activity that is greater than the in vivo activity of apolydispersed mixture of drug-oligomer conjugates having the same numberaverage molecular weight as the mixture of drug-oligomer conjugateswhere each conjugate in the mixture has the same number of polyethyleneglycol subunits. As will be understood by those skilled in the art, thenumber average molecular weight of the mixture of drug-oligomerconjugates where each conjugate in the mixture has the same number ofpolyethylene glycol subunits and the number average weight of thepolydispersed mixture may be measured by various methods including, butnot limited to, size exclusion chromatography such as gel permeationchromatography as described, for example, in H. R. Allcock & F. W.Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).

[0311] As another example, a mixture of drug-oligomer conjugates whereeach conjugate in the mixture has the same number of polyethylene glycolsubunits preferably has an in vitro activity that is greater than the invitro activity of a polydispersed mixture of drug-oligomer conjugateshaving the same number average molecular weight as the mixture ofdrug-oligomer conjugates where each conjugate in the mixture has thesame number of polyethylene glycol subunits. As will be understood bythose skilled in the art, the number average molecular weight of themixture of drug-oligomer conjugates where each conjugate in the mixturehas the same number of polyethylene glycol subunits and the numberaverage weight of the polydispersed mixture may be measured by variousmethods including, but not limited to, size exclusion chromatography.

[0312] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study.

[0313] As still another example, a mixture of drug-oligomer conjugateswhere each conjugate in the mixture has the same number of polyethyleneglycol subunits preferably has an increased resistance to degradation bychymotrypsin when compared to the resistance to degradation bychymotrypsin of a polydispersed mixture of drug-oligomer conjugateshaving the same number average molecular weight as the mixture ofdrug-oligomer conjugates where each conjugate in the mixture has thesame number of polyethylene glycol subunits. As will be understood bythose skilled in the art, the number average molecular weight of themixture of drug-oligomer conjugates where each conjugate in the mixturehas the same number of polyethylene glycol subunits and the numberaverage weight of the polydispersed mixture may be measured by variousmethods including, but not limited to, size exclusion chromatography.

[0314] As yet another example, a mixture of drug-oligomer conjugateswhere each conjugate in the mixture has the same number of polyethyleneglycol subunits preferably has an inter-subject variability that is lessthan the inter-subject variability of a polydispersed mixture ofdrug-oligomer conjugates having the same number average molecular weightas the mixture of drug-oligomer conjugates where each conjugate in themixture has the same number of polyethylene glycol subunits. As will beunderstood by those skilled in the art, the number average molecularweight of the mixture of drug-oligomer conjugates where each conjugatein the mixture has the same number of polyethylene glycol subunits andthe number average weight of the polydispersed mixture may be measuredby various methods including, but not limited to, size exclusionchromatography. The inter-subject variability may be measured by variousmethods as will be understood by those skilled in the art. Theinter-subject variability is preferably calculated as follows. The areaunder a dose response curve (AUC) (i.e., the area between thedose-response curve and a baseline value) is determined for eachsubject. The average AUC for all subjects is determined by summing theAUCs of each subject and dividing the sum by the number of subjects. Theabsolute value of the difference between the subject's AUC and theaverage AUC is then determined for each subject. The absolute values ofthe differences obtained are then summed to give a value that representsthe inter-subject variability. Lower values represent lowerinter-subject variabilities and higher values represent higherinter-subject variabilities.

[0315] Mixtures of drug-oligomer conjugates where each conjugate in themixture has the same number of polyethylene glycol subunits according toembodiments of the present invention preferably have two or more of theabove-described improved properties. More preferably, mixtures ofdrug-oligomer conjugates where each conjugate in the mixture has thesame number of polyethylene glycol subunits according to embodiments ofthe present invention have three or more of the above-described improvedproperties. Most preferably, mixtures of drug-oligomer conjugates whereeach conjugate in the mixture has the same number of polyethylene glycolsubunits according to embodiments of the present invention have all fourof the above-described improved properties.

[0316] According to still other embodiments of the present invention, amixture of conjugates is provided in which each conjugate has the samemolecular weight and has the formula:

[0317] wherein:

[0318] B is a bonding moiety;

[0319] L is a linker moiety;

[0320] G, G′ and G″ are individually selected spacer moieties;

[0321] R is a lipophilic moiety and R′ is a polyalkylene glycol moiety,or R′ is the lipophilic moiety and R is the polyalkylene glycol moiety;

[0322] T is a terminating moiety;

[0323] h, i, j, k, m and n are individually 0 or 1, with the provisothat when R is the polyalkylene glycol moiety; m is 1, and when R′ isthe polyalkylene glycol moiety, n is 1; and

[0324] p is an integer from 1 to the number of nucleophilic residues onthe drug.

[0325] According to these embodiments of the present invention, R or R′is a polyalkylene moiety. The oligomer may be various oligomerscomprising a polyalkylene glycol moiety as will be understood by thoseskilled in the art. Preferably, the polyalkylene glycol moiety has atleast 2, 3, or 4 polyalkylene glycol subunits. More preferably, thepolyalkylene glycol moiety has at least 5 or 6 polyalkylene glycolsubunits. Mo st preferably, the polyalkylene glycol moiety of theoligomer has at least 7 polyalkylene glycol subunits. The polyalkyleneglycol moiety of the oligomer is preferably a lower alkyl polyalkyleneglycol moiety such as a polyethylene glycol moiety, a polypropyleneglycol moiety, or a polybutylene glycol moiety. When the polyalkylenemoiety is a polypropylene glycol moiety, the polypropylene glycol moietypreferably has a uniform structure. An exemplary polypropylene glycolmoiety having a uniform structure is as follows:

[0326] This uniform polypropylene glycol structure may be described ashaving only one methyl substituted carbon atom adjacent each oxygen atomin the polypropylene glycol chain. Such uniform polypropylene glycolmoieties may exhibit both lipophilic and hydrophilic characteristics andthus be useful in providing amphiphilic drug-oligomer conjugates withoutthe use of lipophilic polymer moieties (i.e., the sum of m+n is 1).Furthermore, coupling the secondary alcohol moiety of the polypropyleneglycol moiety with a drug may provide the drug (e.g., a polypeptide)with improved resistance to degradation caused by enzymes such astrypsin and chymotrypsin found, for example, in the gut.

[0327] Uniform polypropylene glycol according to embodiments of thepresent invention is preferably synthesized as illustrated in FIGS. 11through 13, which will now be described. As illustrated in FIG. 11,1,2-propanediol 53 is reacted with a primary alcohol blocking reagent toprovide a secondary alcohol extension monomer 54. The primary alcoholblocking reagent may be various primary alcohol blocking reagents aswill be understood by those skilled in the art including, but notlimited to, silylchloride compounds such as t-butyldiphenylsilylchlorideand t-butyldimethylsilylchloride, and esterification reagents such asAc₂O. Preferably, the primary alcohol blocking reagent is a primaryalcohol blocking reagent that is substantially non-reactive withsecondary alcohols, such as t-butyldiphenylsilylchloride ort-butyldimethylsilylchloride. The secondary alcohol extension monomer(54) may be reacted with methanesulfonyl chloride (MeSO₂Cl) to provide aprimary extension alcohol monomer mesylate 55.

[0328] Alternatively, the secondary alcohol extension monomer 54 may bereacted with a secondary alcohol blocking reagent to provide compound56. The secondary alcohol blocking reagent may be various secondaryalcohol blocking reagents as will be understood by those skilled in theart including, but not limited to, benzyl chloride. The compound 56 maybe reacted with a B₁ de-blocking reagent to remove the blocking moietyB₁ and provide a primary alcohol extension monomer 57. The B₁de-blocking reagent may be selected from various de-blocking reagents aswill be understood by one skilled in the art. When the primary alcoholhas been blocked by forming an ester, the B₁ de-blocking reagent is ade-esterification reagent, such as a base (e.g., potassium carbonate).When the primary alcohol has been blocked using a silylchloride, the B₁de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).The primary alcohol extension monomer 57 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension monomermesylate 58.

[0329] The primary alcohol extension monomer 54 and the secondaryalcohol extension monomer 57 may be capped as follows. The secondaryalcohol extension monomer 54 may be reacted with a capping reagent toprovide a compound 59. The capping reagent may be various cappingreagents as will be understood by those skilled in the art including,but not limited to, alkyl halides such as methyl chloride. The compound59 may be reacted with a B₁ de-blocking agent as described above toprovide a primary alcohol capping monomer 60. The primary alcoholcapping monomer 60 may be reacted with methane sulfonyl chloride toprovide the secondary alcohol capping monomer mesylate 61. The primaryalcohol extension monomer 57 may be reacted with a capping reagent toprovide a compound 62. The capping reagent may be various cappingreagents as described above. The compound 62 may be reacted with a B₂de-blocking reagent to remove the blocking moiety B₂ and provide asecondary alcohol capping monomer 63. The B₂ de-blocking reagent may bevarious de-blocking agents as will be understood by those skilled in theart including, but not limited to, H₂ in the presence of apalladium/activated carbon catalyst. The secondary alcohol cappingmonomer may be reacted with methanesulfonyl chloride to provide aprimary alcohol capping monomer mesylate 64. While the embodimentsillustrated in FIG. 11 show the synthesis of capping monomers, it is tobe understood that similar reactions may be performed to provide cappingpolymers.

[0330] In general, chain extensions may be effected by reacting aprimary alcohol extension mono- or poly-mer such as the primary alcoholextension monomer 57 with a primary alcohol extension mono- or poly-mermesylate such as the primary alcohol extension monomer mesylate 55 toprovide various uniform polypropylene chains or by reacting a secondaryalcohol extension mono- or poly-mer such as the secondary alcoholextension monomer 54 with a secondary alcohol extension mono-or poly-mermesylate such as the secondary alcohol extension monomer mesylate 58.

[0331] For example, in FIG. 13, the primary alcohol extension monomermesylate 55 is reacted with the primary alcohol extension monomer 57 toprovide a dimer compound 65. Alternatively, the secondary alcoholextension monomer mesylate 58 may be reacted with the secondary alcoholextension monomer 54 to provide the dimer compound 65. The B₁ blockingmoiety on the dimer compound 65 may be removed using a B₁ de-blockingreagent as described above to provide a primary alcohol extension dimer66. The primary alcohol extension dimer 66 may be reacted with methanesulfonyl chloride to provide a secondary alcohol extension dimermesylate 67. Alternatively, the B₂ blocking moiety on the dimer compound65 may be removed using the B₂ de-blocking reagent as described above toprovide a secondary alcohol extension dimer 69. The secondary alcoholextension dimer 69 may be reacted with methane sulfonyl chloride toprovide a primary alcohol extension dimer mesylate 70.

[0332] As will be understood by those skilled in the art, the chainextension process may be repeated to achieve various other chainlengths. For example, as illustrated in FIG. 13, the primary alcoholextension dimer 66 may be reacted with the primary alcohol extensiondimer mesylate 70 to provide a tetramer compound 72. As furtherillustrated in FIG. 13, a generic chain extension reaction schemeinvolves reacting the primary alcohol extension mono- or poly-mer 73with the primary alcohol extension mono- or poly-mer mesylate 74 toprovide the uniform polypropylene polymer 75. The values of m and n mayeach range from 0 to 1000 or more. Preferably, m and n are each from 0to 50. While the embodiments illustrated in FIG. 13 show primary alcoholextension mono- and/or poly-mers being reacted with primary alcoholextension mono- and/or poly-mer mesylates, it is to be understood thatsimilar reactions may be carried out using secondary alcohol extensionmono- and/or poly-mers and secondary alcohol extension mono- and/orpoly-mer mesylates.

[0333] An end of a primary alcohol extension mono- or poly-mer or an endof a primary alcohol extension mono- or poly-mer mesylate may be reactedwith a primary alcohol capping mono- or poly-mer mesylate or a primaryalcohol capping mono- or poly-mer, respectively, to provide a cappeduniform polypropylene chain. For example, as illustrated in FIG. 12, theprimary alcohol extension dimer mesylate 70 is reacted with the primaryalcohol capping monomer 60 to provide the capped/blocked primary alcoholextension trimer 71. As will be understood by those skilled in the art,the B₁ blocking moiety may be removed and the resulting capped primaryalcohol extension trimer may be reacted with a primary alcohol extensionmono- or poly-mer mesylate to extend the chain of the capped trimer 71.

[0334] An end of a secondary alcohol extension mono-or poly-mer or anend of a secondary alcohol extension mono-or poly-mer mesylate may bereacted with a secondary alcohol capping mono-or poly-mer mesylate or asecondary alcohol capping mono- or poly-mer, respectively, to provide acapped uniform polypropylene chain. For example, as illustrated in FIG.12, the secondary alcohol extension dimer mesylate 67 is reacted withthe secondary alcohol capping monomer 63 to provide the capped/blockedprimary alcohol extension trimer 68. The B₂ blocking moiety may beremoved as described above and the resulting capped secondary alcoholextension trimer may be reacted with a secondary alcohol extension mermesylate to extend the chain of the capped trimer 68. While thesyntheses illustrated in FIG. 12 show the reaction of a dimer with acapping monomer to provide a trimer, it is to be understood that thecapping process may be performed at any point in the synthesis of auniform polypropylene glycol moiety, or, alternatively, uniformpolypropylene glycol moieties may be provided that are not capped. Whilethe embodiments illustrated in FIG. 12 show the capping of apolybutylene oligomer by synthesis with a capping monomer, it is to beunderstood that polybutylene oligomers of the present invention may becapped directly (i.e., without the addition of a capping monomer) usinga capping reagent as described above in FIG. 11.

[0335] Uniform polypropylene glycol moieties according to embodiments ofthe present invention may be coupled to a drug, a lipophilic moiety suchas a carboxylic acid, and/or various other moieties by various methodsas will be understood by those skilled in the art including, but notlimited to, those described herein with respect to polyethylene glycolmoieties.

[0336] According to these embodiments of the present invention, R or R′is a lipophilic moiety as will be understood by those skilled in theart. The lipophilic moiety is preferably a saturated or unsaturated,linear or branched alkyl moiety or a saturated or unsaturated, linear orbranched fatty acid moiety. When the lipophilic moiety is an alkylmoiety, it is preferably a linear, saturated or unsaturated alkyl moietyhaving 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to12 carbon atoms. When the lipophilic moiety is a fatty acid moiety, itis preferably a natural fatty acid moiety that is linear, saturated orunsaturated, having 2 to 18 carbon atoms. More preferably, the fattyacid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acidmoiety has at least 4, 5 or 6 carbon atoms.

[0337] According to these embodiments of the present invention, thespacer moieties, G, G′ and G″, are spacer moieties as will be understoodby those skilled in the art. Spacer moieties are preferably selectedfrom the group consisting of sugar, cholesterol and glycerine moieties.Preferably, oligomers of these embodiments do not include spacermoieties (i.e., i, j and k are preferably 0).

[0338] According to these embodiments of the present invention, thelinker moiety, L, may be used to couple the oligomer with the drug aswill be understood by those skilled in the art. Linker moieties arepreferably selected from the group consisting of alkyl and fatty acid.

[0339] According to these embodiments of the present invention, theterminating moiety is preferably an alkyl or alkoxy moiety, and is morepreferably a lower alkyl or lower alkoxy moiety. Most preferably, theterminating moiety is methyl or methoxy. While the terminating moiety ispreferably an alkyl or alkoxy moiety, it is to be understood that theterminating moiety may be various moieties as will be understood bythose skilled in the art including, but not limited to, sugars,cholesterol, alcohols, and fatty acids.

[0340] According to these embodiments of the present invention, theoligomer, which is represented by the bracketed portion of the structureof Formula A, is covalently coupled to the drug. In some embodiments,the drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g.,an ester or carbonate bond). A hydrolyzable coupling may provide adrug-oligomer conjugate that acts as a prodrug. In certain instances,for example where the drug-oligomer conjugate is inactive (i.e., theconjugate lacks the ability to affect the body through the drug'sprimary mechanism of action), a hydrolyzable coupling may provide for atime-release or controlled-release effect, administering the drug over agiven time period as one or more oligomers are cleaved from theirrespective drug-oligomer conjugates to provide the active drug. In otherembodiments, the drug is coupled to the oligomer utilizing anon-hydrolyzable bond (e.g., a carbamate, amide, or ether bond). Use ofa non-hydrolyzable bond may be preferable when it is desirable to allowthe drug-oligomer conjugate to circulate in the bloodstream for anextended period of time, preferably at least 2 hours. The bondingmoiety, B, may be various bonding moieties that may be used tocovalently couple the oligomer with the drug as will be understood bythose skilled in the art. Bonding moieties are preferably selected fromthe group consisting of covalent bond(s), ester moieties, carbonatemoieties, carbamate moieties, amide moieties and secondary aminemoieties.

[0341] The variable p is an integer from 1 to the number of nucleophilicresidues on the drug. When p is greater than 1, more than one oligomer(i.e., a plurality of oligomers) is coupled to the drug. According tothese embodiments of the present invention, the oligomers in theplurality are the same.

[0342] The oligomer may be coupled to the drug at various nucleophilicresidues of the drug including, but not limited to, nucleophilichydroxyl finctions and/or amino functions. When the drug is apolypeptide, a nucleophilic hydroxyl function may be found, for example,at serine and/or tyrosine residues, and a nucleophilic amino functionmay be found, for example, at histidine and/or lysine residues, and/orat the one or more N-termini of the polypeptide. When an oligomer iscoupled to the one or more N-termini of the polypeptide, the couplingpreferably forms a secondary amine. For example, when the drug is humaninsulin, the oligomer may be coupled to an amino functionality of theinsulin including the amino functionality of Gly^(A1), the aminofunctionality of Phe^(B1), and the amino functionality of Lys^(B29).When one oligomer is coupled to the human insulin, the oligomer ispreferably coupled to the amino functionality of Lys^(B29). When twooligomers are coupled to the human insulin, the oligomers are preferablycoupled to the amino functionality of Phe^(B1) and the aminofunctionality of Lys^(B29). While more than one oligomer may be coupledto the human insulin, a higher activity (improved glucose loweringability) is observed for the mono-conjugated human insulin. As anotherexample, when the drug is salmon calcitonin, the oligomer may be coupledto an amino functionality of the salmon calcitonin, including the aminofunctionality of Lys¹¹, Lys¹⁸ and the N-terminus. While one or moreoligomers may be coupled to the salmon calcitonin, a higher activity(improved glucose lowering ability) is observed for the di-conjugatedsalmon calcitonin where an oligomer is coupled to the aminofunctionality of Lys¹¹ and an oligomer is coupled to the aminofunctionality of Lys¹⁸. As yet another example, when the drug is humangrowth hormone, the oligomer may be coupled to an amino functionality ofPhe¹, Lys³⁸, Lys⁴¹, Lys⁷⁰, Lys¹¹⁵, Lys¹⁴⁰, Lys¹⁴⁵, Lys¹⁵⁸, Lys¹⁶⁸,and/or Lys¹⁷².

[0343] Mixtures of drug-oligomer conjugates where each conjugate in themixture has the same molecular weight and has the structure of Formula Amay be synthesized by various methods. For example, a mixture ofoligomers consisting of carboxylic acid and polyethylene glycol issynthesized by contacting a mixture of carboxylic acid with a mixture ofpolyethylene glycol under conditions sufficient to provide a mixture ofoligomers. The oligomers of the mixture are then activated so that theyare capable of reacting with a drug to provide a drug-oligomerconjugate. One embodiment of a synthesis route for providing a mixtureof activated oligomers where each oligomer has the same molecular weightand has a structure of the oligomer of Formula A is illustrated in FIG.3 and described in Examples 11-18 hereinbelow. Another embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer has the same molecular weight and has a structure of theoligomer of Formula A is illustrated in FIG. 4 and described in Examples19-24 hereinbelow. Still another embodiment of a synthesis route forproviding a mixture of activated oligomers where each oligomer has thesame molecular weight and has a structure of the oligomer of Formula Ais illustrated in FIG. 5 and described in Examples 25-29 hereinbelow.Yet another embodiment of a synthesis route for providing a mixture ofactivated oligomers where each oligomer has the same molecular weightand has a structure of the oligomer of Formula A is illustrated in FIG.6 and described in Examples 30-31 hereinbelow. Another embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer has the same molecular weight and has a structure of theoligomer of Formula A is illustrated in FIG. 7 and described in Examples32-37 hereinbelow. Still another embodiment of a synthesis route forproviding a mixture of activated oligomers where each oligomer has thesame molecular weight and has a structure of the oligomer of Formula Ais illustrated in FIG. 8 and described in Example 38 hereinbelow. Yetanother embodiment of a synthesis route for providing a mixture ofactivated oligomers where each oligomer has the same molecular weightand has a structure of the oligomer of Formula A is illustrated in FIG.9 and described in Example 39 hereinbelow. Another embodiment of asynthesis route for providing a mixture of activated oligomers whereeach oligomer has the same molecular weight and has a structure of theoligomer of Formula A is illustrated in FIG. 10 and described in Example40 hereinbelow.

[0344] The mixture of activated oligomers where each oligomer has thesame molecular weight and has a structure of the oligomer of Formula Ais reacted with a mixture of drugs where each drug in the mixture hasthe same molecular weight under conditions sufficient to provide amixture of drug-oligomer conjugates, as described, for example, inExamples 41-120 hereinbelow. As will be understood by those skilled inthe art, the reaction conditions (e.g., selected molar ratios, solventmixtures and/or pH) may be controlled such that the mixture ofdrug-oligomer conjugates resulting from the reaction of the mixture ofactivated oligomers where each oligomer has the same molecular weightand has a structure of the oligomer of Formula A and the mixture ofdrugs is a mixture of conjugates where each conjugate has the samemolecular weight and has the structure Formula A. For example,conjugation at the amino finctionality of lysine may be suppressed bymaintaining the pH of the reaction solution below the pK_(a) of lysine.Alternatively, the mixture of drug-oligomer conjugates may be separatedand isolated utilizing, for example, HPLC to provide a mixture ofdrug-oligomer conjugates, for example mono-, di-, or tri-conjugates,where each conjugate in the mixture has the same number molecular weightand has the structure of Formula A. The degree of conjugation (e.g.,whether the isolated molecule is a mono-, di-, or tri-conjugate) of aparticular isolated conjugate may be determined and/or verifiedutilizing various techniques as will be understood by those skilled inthe art including, but not limited to, mass spectroscopy. The particularconjugate structure (e.g., whether the oligomer is at Gly^(A1),Phe^(B1), or Lys^(B29) of a human insulin monoconjugate) may bedetermined and/or verified utilizing various techniques as will beunderstood by those skilled in the art including, but not limited to,sequence analysis, peptide mapping, selective enzymatic cleavage, and/orendopeptidase cleavage.

[0345] As will be understood by those skilled in the art, one or more ofthe reaction sites on the drug may be blocked by, for example, reactingthe drug with a suitable blocking reagent such as N-tert-butoxycarbonyl(t-BOC), or N-(9-fluorenyhnethoxycarbonyl) (N-FMOC). This process may bepreferred, for example, when the drug is a polypeptide and it is desiredto form an unsaturated conjugate (i.e., a conjugate wherein not allnucleophilic residues are conjugated) having an oligomer at the one ormore N-termini of the polypeptide. Following such blocking, the mixtureof blocked drugs may be reacted with the mixture of activated oligomerswhere each oligomer in the mixture has the same molecular weight and hasa structure of the oligomer of Formula A to provide a mixture ofdrug-oligomer conjugates having oligomer(s) coupled to one or morenucleophilic residues and having blocking moieties coupled to othernucleophilic residues. After the conjugation reaction, the drug-oligomerconjugates may be de-blocked as will be understood by those skilled inthe art. If necessary, the mixture of drug-oligomer conjugates may thenbe separated as described above to provide a mixture of drug-oligomerconjugates where each conjugate in the mixture has the same molecularweight and has the structure of Formula A. Alternatively, the mixture ofdrug-oligomer conjugates may be separated prior to de-blocking.

[0346] Mixtures of drug-oligomer conjugates where each conjugate in themixture has the same molecular weight and has the structure of Formula Aaccording to embodiments of the present invention preferably haveimproved properties when compared with those of conventional mixtures.For example, a mixture of drug-oligomer conjugates where each conjugatein the mixture has the same molecular weight and has the structure ofFormula A preferably has an in vivo activity that is greater than the invivo activity of a polydispersed mixture of drug-oligomer conjugateshaving the same number average molecular weight as the mixture ofdrug-oligomer conjugates where each conjugate in the mixture has thesame molecular weight and has the structure of Formula A. As will beunderstood by those skilled in the art, the number average molecularweight of the mixture of drug-oligomer conjugates where each conjugatein the mixture has the same molecular weight and has the structure ofFormula A and the number average weight of the polydispersed mixture maybe measured by various methods including, but not limited to, sizeexclusion chromatography such as gel permeation chromatography asdescribed, for example, in H. R. Allcock & F. W. Lampe, CONTEMPORARYPOLYMER CHEMIsTRY 394-402 (2d. ed., 1991).

[0347] As another example, a mixture of drug-oligomer conjugates whereeach conjugate in the mixture has the same molecular weight and has thestructure of Formula A preferably has an in vitro activity that isgreater than the in vitro activity of a polydispersed mixture ofdrug-oligomer conjugates having the same number average molecular weightas the mixture of drug-oligomer conjugates where each conjugate in themixture has the same molecular weight and has the structure of FormulaA. As will be understood by those skilled in the art, the number averagemolecular weight of the mixture of drug-oligomer conjugates where eachconjugate in the mixture has the same molecular weight and has thestructure of Formula A and the number average weight of thepolydispersed mixture may be measured by various methods including, butnot limited to, size exclusion chromatography.

[0348] The in vitro activity of a particular mixture may be measured byvarious methods, as will be understood by those skilled in the art.Preferably, the in vitro activity is measured using a Cytosensor®Microphysiometer commercially available from Molecular DevicesCorporation of Sunnyvale, Calif. The microphysiometer monitors smallchanges in the rates of extracellular acidification in response to adrug being added to cultured cells in a transwell. This response isproportional to the activity of the molecule under study.

[0349] As still another example, a mixture of drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A preferably has an increased resistance todegradation by chymotrypsin when compared to the resistance todegradation by chymotrypsin of a polydispersed mixture of drug-oligomerconjugates having the same number average molecular weight as themixture of drug-oligomer conjugates where each conjugate in the mixturehas the same molecular weight and has the structure of Formula A. Aswill be understood by those skilled in the art, the number averagemolecular weight of the mixture of drug-oligomer conjugates where eachconjugate in the mixture has the same molecular weight and has thestructure of Formula A and the number average weight of thepolydispersed mixture may be measured by various methods including, butnot limited to, size exclusion chromatography.

[0350] As yet another example, a mixture of drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A preferably has an inter-subjectvariability that is less than the inter-subject variability of apolydispersed mixture of drug-oligomer conjugates having the same numberaverage molecular weight as the mixture of drug-oligomer conjugateswhere each conjugate in the mixture has the same molecular weight andhas the structure of Formula A. As will be understood by those skilledin the art, the number average molecular weight of the mixture ofdrug-oligomer conjugates where each conjugate in the mixture has thesame molecular weight and has the structure of Formula A and the numberaverage weight of the polydispersed mixture may be measured by variousmethods including, but not limited to, size exclusion chromatography.The inter-subject variability may be measured by various methods as willbe understood by those skilled in the art. The inter-subject variabilityis preferably calculated as follows. The area under a dose responsecurve (AUC) (i.e., the area between the dose-response curve and abaseline value) is determined for each subject. The average AUC for allsubjects is determined by summing the AUCs of each subject and dividingthe sum by the number of subjects. The absolute value of the differencebetween the subject's AUC and the average AUC is then determined foreach subject. The absolute values of the differences obtained are thensummed to give a value that represents the inter-subject variability.Lower values represent lower inter-subject variabilities and highervalues represent higher inter-subject variabilities.

[0351] Mixtures of drug-oligomer conjugates where each conjugate in themixture has the same molecular weight and has the structure of Formula Aaccording to embodiments of the present invention preferably have two ormore of the above-described improved properties. More preferably,mixtures of drug-oligomer conjugates where each conjugate in the mixturehas the same molecular weight and has the structure of Formula Aaccording to embodiments of the present invention have three or more ofthe above-described improved properties. Most preferably, mixtures ofdrug-oligomer conjugates where each conjugate in the mixture has thesame molecular weight and has the structure of Formula A according toembodiments of the present invention have all four of theabove-described improved properties.

[0352] Pharmaceutical compositions comprising a conjugate mixtureaccording to embodiments of the present invention are also provided. Themixtures of drug-oligomer conjugates described above may be formulatedfor administration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science And Practice of Pharmacy(9^(th) Ed. 1995). In the manufacture of a pharmaceutical compositionaccording to embodiments of the present invention, the mixture ofdrug-oligomer conjugates is typically admixed with, inter alia, apharmaceutically acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the pharmaceutical composition and should not be deleterious to thepatient. The carrier may be a solid or a liquid, or both, and ispreferably formulated with the mixture of drug-oligomer conjugates as aunit-dose formulation, for example, a tablet, which may contain fromabout 0.01 or 0.5% to about 95% or 99% by weight of the mixture ofdrug-oligomer conjugates.

[0353] The pharmaceutical compositions may be prepared by any of thewell known techniques of pharmacy including, but not limited to,admixing the components, optionally including one or more accessoryingredients.

[0354] The pharmaceutical compositions according to embodiments of thepresent invention include those suitable for oral, rectal, topical,inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal,parenteral (e.g., subcutaneous, intramuscular, intradermal,intraarticular, intrapleural, intraperitoneal, inracerebral,intraarterial, or intravenous), topical (i.e., both skin and mucosalsurfaces, including airway surfaces) and transdermal administration,although the most suitable route in any given case will depend on thenature and severity of the condition being treated and on the nature ofthe particular mixture of drug-oligomer conjugates which is being used.

[0355] Pharmaceutical compositions suitable for oral administration maybe presented in discrete units, such as capsules, cachets, lozenges, ortables, each containing a predetermined amount of the mixture ofdrug-oligomer conjugates; as a powder or granules; as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil emulsion. Such formulations may be prepared by any suitablemethod of pharmacy which includes the step of bringing into associationthe mixture of drug-oligomer conjugates and a suitable carrier (whichmay contain one or more accessory ingredients as noted above). Ingeneral, the pharmaceutical composition according to embodiments of thepresent invention are prepared by uniformly and intimately admixing themixture of drug-oligomer conjugates with a liquid or finely dividedsolid carrier, or both, and then, if necessary, shaping the resultingmixture. For example, a tablet may be prepared by compressing or moldinga powder or granules containing the mixture of drug-oligomer conjugates,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the mixture in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets may be made by molding, in a suitable machine,the powdered compound moistened with an inert liquid binder.

[0356] Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the mixture of drug-oligomerconjugates in a flavoured base, usually sucrose and acacia ortragacanth; and pastilles comprising the mixture of drug-oligomerconjugates in an inert base such as gelatin and glycerin or sucrose andacacia.

[0357] Pharmaceutical compositions according to embodiments of thepresent invention suitable for parenteral administration comprisesterile aqueous and non-aqueous injection solutions of the mixture ofdrug-oligomer conjugates, which preparations are preferably isotonicwith the blood of the intended recipient. These preparations may containanti-oxidants, buffers, bacteriostats and solutes which render thecomposition isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions may include suspending agents andthickening agents. The compositions may be presented in unit\dose ormulti-dose containers, for example sealed ampoules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline orwater-for-injection immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described. For example, aninjectable, stable, sterile composition comprising a mixture ofdrug-oligomer conjugates in a unit dosage form in a sealed container maybe provided. The mixture of drug-oligomer conjugates is provided in theform of a lyophilizate which is capable of being reconstituted with asuitable pharmaceutically acceptable carrier to form a liquidcomposition suitable for injection thereof into a subject. The unitdosage form typically comprises from about 10 mg to about 10 grams ofthe mixture of drug-oligomer conjugates. When the mixture ofdrug-oligomer conjugates is substantially water-insoluble, a sufficientamount of emulsifying agent which is physiologically acceptable may beemployed in sufficient quantity to emulsify the mixture of drug-oligomerconjugates in an aqueous carrier. One such useful emulsifying agent isphosphatidyl choline.

[0358] Pharmaceutical compositions suitable for rectal administrationare preferably presented as unit dose suppositories. These may beprepared by admixing the mixture of drug-oligomer conjugates with one ormore conventional solid carriers, for example, cocoa butter, and thenshaping the resulting mixture.

[0359] Pharmaceutical compositions suitable for topical application tothe skin preferably take the form of an ointment, cream, lotion, paste,gel, spray, aerosol, or oil. Carriers which may be used includepetroleum jelly, lanoline, polyethylene glycols, alcohols, transdermalenhancers, and combinations of two or more thereof.

[0360] Pharmaceutical compositions suitable for transdermaladministration may be presented as discrete patches adapted to remain inintimate contact with the epidermis of the recipient for a prolongedperiod of time. Compositions suitable for transdermal administration mayalso be delivered by iontophoresis (see, for example, PharmaceuticalResearch 3 (6):318 (1986)) and typically take the form of an optionallybuffered aqueous solution of the mixture of drug-oligomer conjugates.Suitable formulations comprise citrate or bis\tris buffer (pH 6) orethanol/water and contain from 0.1 to 0.2M active ingredient.

[0361] Methods of treating an insulin deficiency in a subject in need ofsuch treatment by administering an effective amount of suchpharmaceutical compositions are also provided. The effective amount ofany mixture of drug-oligomer conjugates, the use of which is in thescope of present invention, will vary somewhat from mixture to mixture,and patient to patient, and will depend upon factors such as the age andcondition of the patient and the route of delivery. Such dosages can bedetermined in accordance with routine pharmacological procedures knownto those skilled in the art. As a general proposition, a dosage fromabout 0.1 to about 50 mg/kg will have therapeutic efficacy, with allweights being calculated based upon the weight of the mixture ofdrug-oligomer conjugates. Toxicity concerns at the higher level mayrestrict intravenous dosages to a lower level such as up to about 10mg/kg, with all weights being calculated based upon the weight of theactive base. A dosage from about 10 mg/kg to about 50 mg/kg may beemployed for oral administration. Typically, a dosage from about 0.5mg/kg to 5 mg/kg may be employed for intramuscular injection. Thefrequency of administration is usually one, two, or three times per dayor as necessary to control the condition. Alternatively, thedrug-oligomer conjugates may be administered by continuous infusion. Theduration of treatment depends on the type of insulin deficiency beingtreated and may be for as long as the life of the patient.

[0362] Methods of synthesizing conjugate mixtures according toembodiments of the present invention are also provided. While thefollowing embodiments of a synthesis route are directed to synthesis ofa substantially monodispersed mixture, similar synthesis routes may beutilized for synthesizing other drug-oligomer conjugate mixturesaccording to embodiments of the present invention.

[0363] A substantially monodispersed mixture of polymers comprisingpolyethylene glycol moieties is provided as illustrated in reaction 1:

[0364] R¹ is H or a lipophilic moiety. R¹ is preferably H, alkyl, arylalkyl, an aromatic moiety, a fatty acid moiety, an ester of a fatty acidmoiety, cholesteryl, or adamantyl. R¹ is more preferably H, lower alkyl,or an aromatic moiety. R¹ is most preferably H, methyl, or benzyl.

[0365] In Formula I, n is from 1 to 25. Preferably n is from 1 to 6.

[0366] X⁺ is a positive ion. Preferably X⁺ is any positive ion in acompound, such as a strong base, that is capable of ionizing a hydroxylmoiety on PEG. Examples of positive ions include, but are not limitedto, sodium ions, potassium ions, lithium ions, cesium ions, and thalliumions.

[0367] R² is H or a lipophilic moiety. R² is preferably linear orbranched alkyl, aryl alkyl, an aromatic moiety, a fatty acid moiety, oran ester of a fatty acid moiety. R² is more preferably lower alkyl,benzyl, a fatty acid moiety having 1 to 24 carbon atoms, or an ester ofa fatty acid moiety having 1 to 24 carbon atoms. R² is most preferablymethyl, a fatty acid moiety having 1 to 18 carbon atoms or an ethylester of a fatty acid moiety having 1 to 18 carbon atoms.

[0368] In Formula II, m is from 1 to 25. Preferably m is from 1 to 6.

[0369] Ms is a mesylate moiety (i.e., CH₃S(O₂)—).

[0370] As illustrated in reaction 1, a mixture of compounds having thestructure of Formula I is reacted with a mixture of compounds having thestructure of Formula II to provide a mixture of polymers comprisingpolyethylene glycol moieties and having the structure of Formula III.The mixture of compounds having the structure of Formula I is asubstantially monodispersed mixture. Preferably, at least about 96, 97,98 or 99 percent of the compounds in the mixture of compounds of FormulaI have the same molecular weight, and, more preferably, the mixture ofcompounds of Formula I is a monodispersed mixture. The mixture ofcompounds of Formula II is a substantially monodispersed mixture.Preferably, at least about 96, 97, 98 or 99 percent of the compounds inthe mixture of compounds of Formula II have the same molecular weight,and, more preferably, the mixture of compounds of Formula II is amonodispersed mixture. The mixture of compounds of Formula III is asubstantially monodispersed mixture. Preferably, at least about 96, 97,98 or 99 percent of the compounds in the mixture of compound of FormulaIII have the same molecular weight. More preferably, the mixture ofcompounds of Formula III is a monodispersed mixture.

[0371] Reaction 1 is preferably performed between about 0C and about 40°C., is more preferably performed between about 15° C. and about 35° C.,and is most preferably performed at room temperature (approximately 25°C.).

[0372] Reaction 1 may be performed for various periods of time as willbe understood by those skilled in the art. Reaction 1 is preferablyperformed for a period of time between about 0.25, 0.5 or 0.75 hours andabout 2, 4 or 8 hours.

[0373] Reaction 1 is preferably carried out in an aprotic solvent suchas, but not limited to, N,N-dimethylacetamide (DMA),N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),hexamethylphosphoric triamide, tetrahydrofuran (THF), dioxane, diethylether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane,N-methylpyrollidinone, tetrahydronaphthalene, decahydronaphthalene,1,2-dichlorobenzene, 1,3-dimethyl-2-imidazolidinone, or a mixturethereof. More preferably, the solvent is DMF, DMA or toluene.

[0374] The molar ratio of the compound of Formula I to the compound ofFormula II is preferably greater than about 1:1. More preferably, themolar ratio is at least about 2:1. By providing an excess of thecompounds of Formula I, one can ensure that substantially all of thecompounds of Formula II are reacted, which may aid in the recovery ofthe compounds of Formula III as discussed below.

[0375] Compounds of Formula I are preferably prepared as illustrated inreaction 2:

[0376] R¹ and X⁺ are as described above and the mixture of compounds ofFormula IV is substantially monodispersed; preferably, at least about96, 97, 98 or 99 percent of the compounds in the mixture of compounds ofFormula IV have the same molecular weight; and, more preferably, themixture of compounds of Formula IV is a monodispersed mixture.

[0377] Various compounds capable of ionizing a hydroxyl moiety on thePEG moiety of the compound of Formula IV will be understood by thoseskilled in the art. The compound capable of ionizing a hydroxyl moietyis preferably a strong base. More preferably, the compound capable ofionizing a hydroxyl moiety is selected from the group consisting ofsodium hydride, potassium hydride, sodium t-butoxide, potassiumt-butoxide, butyl lithium (BuLi), and lithium diisopropylamine. Thecompound capable of ionizing a hydroxyl moiety is more preferably sodiumhydride.

[0378] The molar ratio of the compound capable of ionizing a hydroxylmoiety on the PEG moiety of the compound of Formula IV to the compoundof Formula IV is preferably at least about 1:1, and is more preferablyat least about 2:1. By providing an excess of the compound capable ofionizing the hydroxyl moiety, it is assured that substantially all ofthe compounds of Formula IV are reacted to provide the compounds ofFormula I. Thus, separation difficulties, which may occur if bothcompounds of Formula IV and compounds of Formula I were present in thereaction product mixture, may be avoided.

[0379] Reaction 2 is preferably performed between about 0° C. and about40° C., is more preferably performed between about 0° C. and about 35°C., and is most preferably performed between about 0° C. and roomtemperature (approximately 25° C.).

[0380] Reaction 2 may be performed for various periods of time as willbe understood by those skilled in the art. Reaction 2 is preferablyperformed for a period of time between about 0.25, 0.5 or 0.75 hours andabout 2, 4 or 8 hours.

[0381] Reaction 2 is preferably carried out in an aprotic solvent suchas, but not limited to, N,N-dimethylacetamide (DMA),N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),hexamethylphosphoric triamide, tetrahydrofuran (THF), dioxane, diethylether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane,N-methylpyrollidinone, dichloromethane, chloroform,tetrahydronaphthalene, decahydronaphthalene, 1,2-dichlorobenzene,1,3-dimethyl-2-imidazolidinone, or a mixture thereof. More preferably,the solvent is DMF, dichloromethane or toluene.

[0382] Compounds of Formula II are preferably prepared as illustrated inreaction 3:

[0383] R² and Ms are as described above and the compound of Formula V ispresent as a substantially monodispersed mixture of compounds of FormulaV; preferably at least about 96, 97, 98 or 99 percent of the compoundsin the mixture of compounds of Formula V have the same molecular weight;and, more preferably, the mixture of compounds of Formula V is amonodispersed mixture.

[0384] Q is a halide, preferably chloride or fluoride.

[0385] CH₃S(O₂)Q is methanesulfonyl halide. The methanesulfonyl halideis preferably methanesulfonyl chloride or methanesulfonyl fluoride. Morepreferably, the methanesulfonyl halide is methanesulfonyl chloride.

[0386] The molar ratio of the methane sulfonyl halide to the compound ofFormula V is preferably greater than about 1:1, and is more preferablyat least about 2:1. By providing an excess of the methane sulfonylhalide, it is assured that substantially all of the compounds of FormulaV are reacted to provide the compounds of Formula II. Thus, separationdifficulties, which may occur if both compounds of Formula V andcompounds of Formula II were present in the reaction product mixture,may be avoided.

[0387] Reaction 3 is preferably performed between about −10° C. andabout 40° C., is more preferably performed between about 0° C. and about35° C., and is most preferably performed between about 0° C. and roomtemperature (approximately 25° C.).

[0388] Reaction 3 may be performed for various periods of time as willbe understood by those skilled in the art. Reaction 3 is preferablyperformed for a period of time between about 0.25, 0.5 or 0.75 hours andabout 2, 4 or 8 hours.

[0389] Reaction 3 is preferably carried out in the presence of analiphatic amine including, but not limited to, monomethylamine,dimethylamine, trimethylamine, monoethylamine, diethylamine,triethylamine, monoisopropylamine, diisopropylamine, mono-n-butylamine,di-n-butylamine, tri-n-butylamine, monocyclohexylamine,dicyclohexylamine, or mixtures thereof. More preferably, the aliphaticamine is a tertiary amine such as triethylamine.

[0390] As will be understood by those skilled in the art, varioussubstantially monodispersed mixtures of compounds of Formula V arecommercially available. For example, when R₂ is H or methyl, thecompounds of Formula V are PEG or mPEG compounds, respectively, whichare commercially available from Aldrich of Milwaukee, Wis.; Fluka ofSwitzerland, and/or TCl America of Portland, Oreg.

[0391] When R² is a lipophilic moiety such as, for example, higheralkyl, fatty acid, an ester of a fatty acid, cholesteryl, or adamantyl,the compounds of Formula V may be provided by various methods as will beunderstood by those skilled in the art. The compounds of Formula V arepreferably provided as follows:

[0392] R² is a lipophilic moiety, preferably higher alkyl, fatty acidester, cholesteryl, or adamantyl, more preferably a lower alkyl ester ofa fatty acid, and most preferably an ethyl ester of a fatty acid havingfrom 1 to 18 carbon atoms.

[0393] R³ is H, benzyl, trityl, tetrahydropyran, or other alcoholprotecting groups as will be understood by those skilled in the art.

[0394] X₂ ⁺ is a positive ion as described above with respect to X⁺.

[0395] The value of m is as described above.

[0396] Regarding reaction 4, a mixture of compounds of Formula VI isreacted with a mixture of compounds of Formula VII under reactionconditions similar to those described above with reference toreaction 1. The mixture of compounds of Formula VI is a substantiallymonodispersed mixture. Preferably, at least about 96, 97, 98 or 99percent of the compounds in the mixture of compounds of Formula VI havethe same molecular weight. More preferably, the mixture of compounds ofFormula VI is a monodispersed mixture. The mixture of compounds ofFormula VII is a substantially monodispersed mixture. Preferably, atleast about 96, 97, 98 or 99 percent of the compounds in the mixture ofcompounds of Formula VII have the same molecular weight. Morepreferably, the mixture of compounds of Formula VII is a monodispersedmixture.

[0397] Regarding reaction 5, the compound of Formula VIII may behydrolyzed to convert the R³ moiety into an alcohol by various methodsas will be understood by those skilled in the art. When R³ is benzyl ortrityl, the hydrolysis is preferably performed utilizing H₂ in thepresence of a palladium-charcoal catalyst as is known by those skilledin the art. Of course, when R³ is H, reaction 5 is unnecessary.

[0398] The compound of Formula VI may be commercially available or beprovided as described above with reference to reaction 3. The compoundof Formula VII may be provided as described above with reference toreaction 2.

[0399] Substantially monodispersed mixtures of polymers comprising PEGmoieties and having the structure of Formula III above can further bereacted with other substantially monodispersed polymers comprising PEGmoieties in order to extend the PEG chain. For example, the followingscheme may be employed:

[0400] Ms, m and n are as described above with reference to reaction 1;p is similar to n and m, and X₂ ⁺ is similar to X⁺ as described abovewith reference to reaction 1. Q is as described above with reference toreaction 3. R² is as described above with reference to reaction 1 and ispreferably lower alkyl. R¹ is H. Reaction 6 is preferably performed in amanner similar to that described above with reference to reaction 3.Reaction 7 is preferably performed in a manner similar to that describedabove with reference to reaction 1. Preferably, at least about 96, 97,98 or 99 percent of the compounds in the mixture of compounds of FormulaIII have the same molecular weight, and, more preferably, the mixture ofcompounds of Formula III is a monodispersed mixture. The mixture ofcompounds of Formula X is a substantially monodispersed mixture.Preferably, at least about 96, 97, 98 or 99 percent of the compounds inthe mixture of compounds of Formula X have the same molecular weight,and, more preferably, the mixture of compounds of Formula X is amonodispersed mixture.

[0401] A process according to embodiments of the present invention isillustrated by the scheme shown in FIG. 1, which will now be described.The synthesis of substantially monodispersed polyethyleneglycol-containing oligomers begins by the preparation of the monobenzylether (1) of a substantially monodispersed polyethylene glycol. Anexcess of a commercially available substantially monodispersedpolyethylene glycol is reacted with benzyl chloride in the presence ofaqueous sodium hydroxide as described by Coudert et al (SyntheticCommunications, 16(1): 19-26 (1986)). The sodium salt of 1 is thenprepared by the addition of NaH, and this sodium salt is allowed toreact with the mesylate synthesized from the ester of a hydroxyalkanoicacid (2). The product (3) of the displacement of the mesylate isdebenzylated via catalytic hydrogenation to obtain the alcohol (4). Themesylate (5) of this alcohol may be prepared by addition ofmethanesulfonyl chloride and used as the electrophile in the reactionwith the sodium salt of the monomethyl ether of a substantiallymonodispersed polyethylene glycol derivative, thereby extending thepolyethylene glycol portion of the oligomer to the desired length,obtaining the elongated ester (6). The ester may be hydrolyzed to theacid (7) in aqueous base and transformed into the activated ester (8) byreaction with a carbodiimide and N-hydroxysuccinimide. While theoligomer illustrated in FIG. 1 is activated using N-hydroxysuccinimide,it is to be understood that various other reagents may be used toactivate oligomers of the present invention including, but not limitedto, active phenyl chloroformates such aspara-nitrophenyl chloroformate,phenyl chloroformate, 3,4-phenyldichloroformate, and3,4-phenyldichloroformate; tresylation; and acetal formation.

[0402] Still referring to FIG. 1, q is from 1 to 24. Preferably, q isfrom 1 to 18, and q is more preferably from 4 to 16. R⁴ is a moietycapable of undergoing hydrolysis to provide the carboxylic acid. R⁴ ispreferably lower alkyl and is more preferably ethyl. The variables n andm are as described above with reference to reaction 1.

[0403] All starting materials used in the procedures described hereinare either commercially available or can be prepared by methods known inthe art using commercially available starting materials.

[0404] The present invention will now be described with reference to thefollowing examples. It should be appreciated that these examples are forthe purposes of illustrating aspects of the present invention, and donot limit the scope of the invention as defined by the claims.

EXAMPLES Examples 1 through 10

[0405] Reactions in Examples 1 through 10 were carried out undernitrogen with magnetic stirring, unless otherwise specified. “Work-up”denotes extraction with an organic solvent, washing of the organic phasewith saturated NaCl solution, drying (MgSO₄), and evaporation (rotaryevaporator). Thin layer chromatography was conducted with Merck glassplates precoated with silica gel 60° F.-254 and spots were visualized byiodine vapor. All mass spectra were determined by MacromolecularResources Colorado State University, Colo. and are reported in the orderm/z, (relative intensity). Elemental analyses and melting points wereperformed by Galbraith Laboratories, Inc., Knoxville, Tenn. Examples1-10 refer to the scheme illustrated in FIG. 2.

Example 1 8-Methoxy-1-(methylsulfonyl)oxy-3,6-dioxaoctane (9)

[0406] A solution of non-polydispersed triethylene glycol monomethylether molecules (4.00 mL, 4.19 g, 25.5 mmol) and triethylamine (4.26 mL,3.09 g, 30.6 mmol) in dry dichloromethane (50 mL) was chilled in an icebath and place under a nitrogen atmosphere. A solution ofmethanesulfonyl chloride (2.37 mL, 3.51 g, 30.6 mmol) in drydichloromethane (20 mL) was added dropwise from an addition funnel. Tenminutes after the completion of the chloride addition, the reactionmixture was removed from the ice bath and allowed to come to roomtemperature. The mixture was stirred for an additional hour, at whichtime TLC (CHCl₃ with 15% MeOH as the elutant) showed no remainingtriethylene glycol monomethyl ether.

[0407] The reaction mixture was diluted with another 75 mL ofdichloromethane and washed successively with saturated NaHCO₃, water andbrine. The organics were dried over Na₂SO₄, filtered and concentrated invacuo to give a non-polydispersed mixture of compounds 9 as a clear oil(5.31 g, 86%).

Example 2 Ethylene glycol mono methyl ether (10) (m=4,5,6)

[0408] To a stirred solution of non-polydispersed compound 11 (35.7mmol) in dry DMF (25.7 mL), under N₂ was added in portion a 60%dispersion of NaH in mineral oil, and the mixture was stirred at roomtemperature for 1 hour. To this salt 12 was added a solution ofnon-polydispersed mesylate 9 (23.36) in dry DMF (4 ml) in a singleportion, and the mixture was stirred at room temperature for 3.5 hours.Progress of the reaction was monitored by TLC (12% CH₃OH—CHCl₃). Thereaction mixture was diluted with an equal amount of 1N HCl, andextracted with ethyl acetate (2×20 ml) and discarded. Extraction ofaqueous solution and work-up gave non-polydispersed polymer 10 (82-84%yield).

Example 3 3,6,9,12,15,18,21-Heptaoxadocosanol (10) (m=4)

[0409] Oil; Rf 0.46 (methanol: chloroform=3:22); MS m/z calc'd forC₁₅H₃₂O₈ 340.21 (M⁺+1), found 341.2.

Example 4 3,6,9,12,15,18,21,24-Octaoxapentacosanol (10) (m=5)

[0410] Oil; Rf 0.43 (methanol: chloroform=6:10); MS m/z calc'd forC₁₇H₃₆O₉ 384.24 (M⁺+1), found 385.3.

Example 5 3,6,9,12,15,18,21,24,27-Nonaoxaoctacosanol (10) (m=6)

[0411] Oil; Rf 0.42 (methanol: chloroform=6:10); MS m/z calc'd forC₁₉H₄₀O₁₀ 428.26 (M⁺+1), found 429.3.

Example 620-methoxy-1-(methylsulfonyl)oxy-3,6,9,12,15,18-hexaoxaeicosane (14)

[0412] Non-polydispersed compound 14 was obtained in quantitative yieldfrom the alcohol 13 (m=4) and methanesulfonyl chloride as described for9, as an oil; Rf 0.4 (ethyl acetate: acetonitrile=1:5); MS m/z calc'dfor C₁₇H₃₇O₁₀ 433.21 (M⁺+1), found 433.469.

Example 7 Ethylene glycol mono methyl ether (15) (m=3,4,5)

[0413] The non-polydispersed compounds 15 were prepared from a diol byusing the procedure described above for compound 10.

Example 8 3,6,9,12,15,18,21,24,27,30-Decaoxaheneicosanol (15) (m=3)

[0414] Oil; Rf 0.41 (methanol: chloroform=6:10); MS m/z calc'd forC₂₁H₄₄O₁₁ 472.29 (M⁺+1), found 472.29.

Example 9 3,6,9,12,15,18,21,24,27,30,33-Unecaoxatetratricosanol (15)(m=4)

[0415] Oil; Rf 0.41 (methanol: chloroform=6:10); MS m/z calc'd forC₂₃H₄₈O₁₂ 516.31 (M⁺+1), found 516.31.

Example 10 3,6,9,12,15,18,21,24,27,30,33,36-Dodecaoxaheptatricosanol(15) (m=5)

[0416] Oil; Rf 0.41 (methanol: chloroform=6: 10); MS m/z calc'd forC₂₅H_(52; O) ₁₃ 560.67 M⁺+1), found 560.67.

[0417] Examples 11 through 18 refer to the scheme illustrated in FIG. 3.

Example 11 Hexaethylene glycol monobenzyl ether (16)

[0418] An aqueous sodium hydroxide solution prepared by dissolving 3.99g (100 mmol) NaOH in 4 ml water was added slowly to non-polydispersedhexaethylene glycol (28.175 g, 25 ml, 100 mmol). Benzyl chloride (3.9 g,30.8 mmol, 3.54 ml) was added and the reaction mixture was heated withstirring to 100° C. for 18 hours. The reaction mixture was then cooled,diluted with brine (250 ml) and extracted with methylene chloride (200ml×2). The combined organic layers were washed with brine once, driedover Na₂SO₄, filtered and concentrated in vacuo to a dark brown oil. Thecrude product mixture was purified via flash chromatography (silica gel,gradient elution: ethyl acetate to 9/1 ethyl acetate/methanol) to yield8.099 g (70 %) of non-polydispersed 16 as a yellow oil.

Example 12 Ethyl 6-methylsulfonyloxyhexanoate (17)

[0419] A solution of non-polydispersed ethyl 6-hydroxyhexanoate (50.76ml, 50.41 g, 227 mmol) in dry dichloromethane (75 ml) was chilled in aice bath and placed under a nitrogen atmosphere. Triethylamine (34.43ml, 24.99 g, 247 mmol) was added. A solution of methanesulfonyl chloride(19.15 ml, 28.3 g, 247 mmol) in dry dichloromethane (75 ml) was addeddropwise from an addition funnel. The mixture was stirred for three andone half hours, slowly being allowed to come to room temperature as theice bath melted. The mixture was filtered through silica gel, and thefiltrate was washed successively with water, saturated NaHCO₃, water andbrine. The organics were dried over Na₂SO₄, filtered and concentrated invacuo to a pale yellow oil. Final purification of the crude product wasachieved by flash chromatography (silica gel, 1/1 hexanes/ethyl acetate)to give the non-polydispersed product (46.13 g, 85%) as a clear,colorless oil. FAB MS: m/e 239 (M+H), 193 (M—C₂H₅O).

Example 136-{2-[2-(2-{2-[2-(2-Benzyloxyethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]-ethoxy}-hexanoicacid ethyl ester (18)

[0420] Sodium hydride (3.225 g or a 60% oil dispersion, 80.6 mmol) wassuspended in 80 ml of anhydrous toluene, placed under a nitrogenatmosphere and cooled in an ice bath. A solution of thenon-polydispersed alcohol 16 (27.3 g, 73.3 mmol) in 80 ml dry toluenewas added to the NaH suspension. The mixture was stirred at 0° C. forthirty minutes, allowed to come to room temperature and stirred foranother five hours, during which time the mixture became a clear brownsolution. The non-polydispersed mesylate 17 (19.21 g, 80.6 mmol) in 80ml dry toluene was added to the NaH/alcohol mixture, and the combinedsolutions were stirred at room temperature for three days. The reactionmixture was quenched with 50 ml methanol and filtered through basicalumina. The filtrate was concentrated in vacuo and purified by flashchromatography (silica gel, gradient elution: 3/1 ethyl acetate/hexanesto ethyl acetate) to yield the non-polydispersed product as a paleyellow oil (16.52 g, 44%). FAB MS: m/e 515 (M+H).

Example 146-{2-[2-(2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]-ethoxy}-hexanoicacid ethyl ester (19)

[0421] Non-polydispersed benzyl ether 18 (1.03 g, 2.0 mmol) wasdissolved in 25 ml ethanol. To this solution was added 270 mg 10% Pd/C,and the mixture was placed under a hydrogen atmosphere and stirred forfour hours, at which time TLC showed the complete disappearance of thestarting material. The reaction mixture was filtered through Celite 545to remove the catalyst, and the filtrate was concentrated in vacuo toyield the non-polydispersed title compound as a clear oil (0.67 g, 79%).FAB MS: m/e 425 (M+H), 447 (M+Na).

Example 156-{2-[2-(2-{2-[2-(2-methylsulfonylethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]-ethoxy}-hexanoicacid ethyl ester (20)

[0422] The non-polydispersed alcohol 19 (0.835 g, 1.97 mmol) wasdissolved in 3.5 ml dry dichloromethane and placed under a nitrogenatmosphere. Triethylamine (0.301 ml, 0.219 g, 2.16 mmol) was added andthe mixture was chilled in an ice bath. After two minutes, themethanesulfonyl chloride (0.16 ml, 0.248 g, 2.16 mmol) was added. Themixture was stirred for 15 minutes at 0° C., then at room temperaturefor two hours. The reaction mixture was filtered through silica gel toremove the triethylammonium chloride, and the filtrate was washedsuccessively with water, saturated NaHCO₃, water and brine. The organicswere dried over Na₂SO₄, filtered and concentrated in vacuo. The residuewas purified by column chromatography (silica gel, 9/1 ethylacetate/methanol) to give non-polydispersed compound 20 as a clear oil(0.819 g, 83%). FAB MS: m/e 503 (M+H).

Example 166-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoicacid ethyl ester (21)

[0423] NaH (88 mg of a 60% dispersion in oil, 2.2 mmol) was suspended inanhydrous toluene (3 ml) under N₂ and chilled to 0° C. Non-polydisperseddiethylene glycol monomethyl ether (0.26 ml, 0.26 g, 2.2 mmol) that hadbeen dried via azeotropic distillation with toluene was added. Thereaction mixture was allowed to warm to room temperature and stirred forfour hours, during which time the cloudy grey suspension became clearand yellow and then turned brown. Mesylate 20 (0.50 g, 1.0 mmol) in 2.5ml dry toluene was added. After stirring at room temperature over night,the reaction was quenched by the addition of 2 ml of methanol and theresultant solution was filtered through silica gel. The filtrate wasconcentrated in vacuo and the FAB MS: m/e 499 (M+H), 521 (M+Na).Additional purification by preparatory chromatography (silica gel, 19/3chloroform/methanol) provided the non-polydispersed product as a clearyellow oil (0.302 g 57%). FAB MS: m/e 527 (M+H), 549 (M+Na).

Example 176-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoicacid (22)

[0424] Non-polydispersed ester 21 (0.25 g, 0.46 mmol) was stirred for 18hours in 0.71 ml of 1 N NaOH. After 18 hours, the mixture wasconcentrated in vacuo to remove the alcohol and the residue dissolved ina further 10 ml of water. The aqueous solution was acidified to pH 2with 2 N HCl and the product was extracted into dichloromethane (30ml×2). The combined organics were then washed with brine (25 ml×2),dried over Na₂SO₄, filtered and concentrated in vacuo to yield thenon-polydispersed title compound as a yellow oil (0.147 g, 62%). FAB MS:mle 499 (M+H), 521 (M+Na).

Example 186-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoicacid 2,5-dioxo-pyrrolidin-1-yl ester (23)

[0425] Non-polydispersed acid 22 (0.209 g, 0.42 mmol) were dissolved in4 ml of dry dichloromethane and added to a dry flask already containingNHS (N-hydroxysuccinimide) (57.8 mg, 0.502 mmol) and EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (98.0 mg,0.502 mmol) under a N₂ atmosphere. The solution was stirred at roomtemperature overnight and filtered through silica gel to remove excessreagents and the urea formed from the EDC. The filtrate was concentratedin vacuo to provide the non-polydispersed product as a dark yellow oil(0.235 g, 94%). FAB MS: mle 596 (M+H), 618 (M+Na).

[0426] Examples 19 through 24 refer to the scheme illustrated in FIG. 4.

Example 19 Mesylate of triethylene glycol monomethyl ether (24)

[0427] To a solution of CH₂Cl₂ (100 mL) cooled to 0° C. in an ice bathwas added non-polydispersed triethylene glycol monomethyl ether (25 g,0.15 mol). Then triethylamine (29.5 mL, 0.22 mol) was added and thesolution was stirred for 15 min at 0° C., which was followed by dropwiseaddition of methanesulfonyl chloride (13.8 mL, 0.18 mol, dissolved in 20mL CH₂Cl₂). The reaction mixture was stirred for 30 min at 0° C.,allowed to warm to room temperature, and then stirred for 2 h. The crudereaction mixture was filtered through Celite (washed CH₂Cl₂˜200 mL),then washed with H₂O (300 mL), 5% NaHCO₃ (300 mL), H₂O (300 mL), sat.NaCl (300 mL), dried MgSO₄, and evaporated to dryness. The oil was thenplaced on a vacuum line for ˜2h to ensure dryness and afforded thenon-polydispersed title compound as a yellow oil (29.15 g, 80% yield).

Example 20 Heptaethylene glycol monomethyl ether (25)

[0428] To a solution of non-polydispersed tetraethylene glycol (51.5 g,0.27 mol) in THF (1L) was added potassium t-butoxide (14.8 g, 0.13 mol,small portions over ˜30 min). The reaction mixture was then stirred for1 h and then 24 (29.15 g, 0.12 mol) dissolved in THF (90 mL) was addeddropwise and the reaction mixture was stirred overnight. The crudereaction mixture was filtered through Celite (washed CH₂Cl₂, ˜200 mL)and evaporated to dryness. The oil was then dissolved in HCl (250 mL, 1N) and washed with ethyl acetate (250 mL) to remove excess 24.Additional washings of ethyl acetate (125 mL) may be required to removeremaining 24. The aqueous phase was washed repetitively with CH₂Cl₂ (125mL volumes) until most of the 25 has been removed from the aqueousphase. The first extraction will contain 24, 25, and dicoupled sideproduct and should be back extracted with HCl (125 mL, 1N). The organiclayers were combined and evaporated to dryness. The resultant oil wasthen dissolved in CH₂Cl₂ (100 mL) and washed repetitively with H20 (50mL volumes) until 25 was removed. The aqueous fractions were combined,total volume 500 mL, and NaCl was added until the solution became cloudyand then was washed with CH₂Cl₂ (2×500 mL). The organic layers werecombined, dried MgSO₄, and evaporated to dryness to afford a thenon-polydispersed title compound as an oil (16.9 g, 41% yield). It maybe desirable to repeat one or more steps of the purification procedureto ensure high purity.

Example 21 8-Bromooctoanate (26)

[0429] To a solution of 8-bromooctanoic acid (5.0 g, 22 mmol) in ethanol(100 mL) was added H₂SO₄ (0.36 mL, 7.5 mmol) and the reaction was heatedto reflux with stirring for 3 h. The crude reaction mixture was cooledto room temperature and washed H₂O (100 mL), sat. NaHCO₃ (2×100 mL), H₂O(100 mL), dried MgSO₄, and evaporated to dryness to afford a clear oil(5.5 g, 98% yield).

Example 22 Synthesis of MPEG7-C8 ester (27)

[0430] To a solution of the non-polydispersed compound 25 (3.0 g, 8.8mmol) in ether (90 mL) was added potassium t-butoxide (1.2 g, 9.6 mmol)and the reaction mixture was stirred for 1 h. Then dropwise addition ofthe non-polydispersed compound 26 (2.4 g, 9.6 mmol), dissolved in ether(10 mL), was added and the reaction mixture was stirred overnight. Thecrude reaction mixture was filtered through Celite (washed CH₂Cl₂, ˜200mL) and evaporated to dryness. The resultant oil was dissolved in ethylacetate and washed H₂O (2×200 mL), dried MgSO₄, and evaporated todryness. Column chromatography (Silica, ethyl acetate to ethylacetate/methanol, 10:1) was performed and afforded the non-polydispersedtitle compound as a clear oil (0.843 g, 19% yield).

Example 23 MPEG7-C8 acid (28)

[0431] To the oil of the non-polydispersed compound 27 (0.70 g, 1.4mmol) was added 1N NaOH (2.0 mL) and the reaction mixture was stirredfor 4 h. The crude reaction mixture was concentrated, acidified (pH˜2),saturated with NaCl, and washed CH₂Cl₂ (2×50 mL). The organic layerswere combined, washed sat. NaCl, dried MgSO₄, and evaporated to drynessto afford the non-polydispersed title compound as a clear oil (0.35 g,53% yield).

Example 24 Activation of MPEG7-C8 acid (29)

[0432] Non-polydispersed mPEG7-C8-acid 28 (0.31 g, 0.64 mmol) wasdissolved in 3 ml of anhydrous methylene chloride and then solution ofN-hydroxysuccinimide (0.079g, 0.69 nimol) and EDCI·HCl (135.6 mg, 0.71mmol) in anhydrous methylene chloride added. Reaction was stirred forseveral hours, then washed with 1N HCl, water, dried over MgSO₄,filtered and concentrated. Crude material was purified by columnchromatography, concentrated to afford the non-polydispersed titlecompound as a clear oil and dried via vacuum.

[0433] Examples 25 through 29 refer to the scheme illustrated in FIG. 5.

Example 25 10-hydroxydecanoate (30)

[0434] To a solution of non-polydispersed 10-hydroxydecanoic acid (5.0g, 26.5 mmol) in ethanol (100 mL) was added H₂SO₄ (0.43 mL, 8.8 mmol)and the reaction was heated to reflux with stirring for 3 h. The crudereaction mixture was cooled to room temperature and washed H₂O (100 mL),sat. NaHCO₃ (2×100 mL), H₂O (100 mL), dried MgSO₄, and evaporated todryness to afford the non-polydispersed title compound as a clear oil(6.9 g, 98% yield).

Example 26 Mesylate of 10-hydroxydecanoate (31)

[0435] To a solution of CH₂Cl₂ (27 mL) was added non-polydispersed10-hydroxydecanoate 30 (5.6 g, 26 mmol) and cooled to 0° C. in an icebath. Then triethylamine (5 mL, 37 mmol) was added and the reactionmixture was stirred for 15 min at 0° C. Then methanesulfonyl chloride(2.7 mL, 24 mmol) dissolved in CH₂Cl₂ (3 mL) was added and the reactionmixture was stirred at 0° C. for 30 min, the ice bath was removed andthe reaction was stirred for an additional 2 h at room temperature. Thecrude reaction mixture was filtered through Celite (washed CH₂Cl₂, 80mL) and the filtrate was washed H₂O (100 mL), 5% NaHCO₃ (2×100 mL), H₂O(100 mL), sat. NaCl (100 mL), dried MgSO₄, and evaporated to dryness toafford the non-polydispersed title compound as a yellowish oil (7.42 g,97% yield).

Example 27 MPEG₇-C₁₀ Ester (32)

[0436] To a solution of non-polydispersed heptaethylene glycolmonomethyl ether 25 (2.5 g, 7.3 mmol) in tetrahydrofuran (100 mL) wasadded sodium hydride (0.194 g, 8.1 mmol) and the reaction mixture wasstirred for 1 h. Then dropwise addition of mesylate of non-polydispersed10-hydroxydecanoate 31 (2.4 g, 8.1 mmol), dissolved in tetrahydrofuran(10 mL), was added and the reaction mixture was stirred overnight. Thecrude reaction mixture was filtered through Celite (washed CH₂Cl₂, ˜200mL) and evaporated to dryness. The resultant oil was dissolved in ethylacetate and washed H₂O (2×200 mL), dried MgSO₄, evaporated to dryness,chromatographed (silica, ethyl acetate/methanol, 10:1), andchromatographed (silica, ethyl acetate) to afford the non-polydispersedtitle compound as a clear oil (0.570 g, 15% yield).

Example 28 MPEG₇-C₁₀ Acid (33)

[0437] To the oil of non-polydispersed mPEG₇-C₁₀ ester 32 (0.570 g, 1.1mmol) was added 1N NaOH (1.6 mL) and the reaction mixture was stirredovernight. The crude reaction mixture was concentrated, acidified(pH˜2), saturated with NaCl, and washed CH₂Cl₂ (2×50 mL). The organiclayers were combined, washed sat. NaCl (2×50 mL), dried MgSO₄, andevaporated to dryness to afford the non-polydispersed title compound asa clear oil (0.340 g, 62% yield).

Example 29 Activation of MPEG₇-C₁₀ Acid (34)

[0438] The non-polydispersed acid 33 was activated using proceduressimilar to those described above in Example 24.

[0439] Examples 30 and 31 refer to the scheme illustrated in FIG. 6.

Example 30 Synthesis of C18(PEG6) Oligomer (36)

[0440] Non-polydispersed stearoyl chloride 35 (0.7 g, 2.31 mmol) wasadded slowly to a mixture of PEG6 (5 g, 17.7 mmol) and pyridine (0.97 g,12.4 mmol) in benzene. The reaction mixture was stirred for severalhours (˜5). The reaction was followed by TLC using ethylacetate/methanolas a developing solvent. Then the reaction mixture was washed withwater, dried over MgSO₄, concentrated and dried via vacuum. Purifiednon-polydispersed compound 36 was analyzed by FABMS: m/e 549/ M⁺H.

Example 31 Activation of C18(PEG6) Oligomer

[0441] Activation of non-polydispersed C18(PEG6) oligomer wasaccomplished in two steps:

[0442] 1) Non-polydispersed stearoyl-PEG636 (0.8 g, 1.46 mmol ) wasdissolved in toluene and added to a phosgene solution (10 ml, 20 % intoluene) which was cooled with an ice bath. The reaction mixture wasstirred for 1 h at 0° C. and then for 3 h at room temperature. Thenphosgene and toluene were distilled off and the remainingnon-polydispersed stearoyl PEG6 chloroformate 37 was dried over P₂O₅overnight.

[0443] 2) To a solution of non-polydispersed stearoyl PEG6 chloroformate36 (0.78 g, 1.27 mmol) and TEA (128 mg, 1.27 mmol) in anhydrousmethylene chloride, N-hydroxy succinimide (NHS) solution in methylenechloride was added. The reaction mixture was stirred for 16 hours, thenwashed with water, dried over MgSO₄, filtered, concentrated and driedvia vacuum to provide the non-polydispersed activated C18(PEG6) oligomer38.

[0444] Examples 32 through 37 refer to the scheme illustrated in FIG. 7.

Example 32 Tetraethylene glycol monobenzylether (39)

[0445] To the oil of non-polydispersed tetraethylene glycol (19.4 g,0.10 mol) was added a solution of NaOH (4.0 g in 4.0 mL) and thereaction was stirred for 15 mm. Then benzyl chloride (3.54 mL, 30.8mmol) was added and the reaction mixture was heated to 100° C. andstirred overnight. The reaction mixture was cooled to room temperature,diluted with sat. NaCl (250 mL), and washed CH₂Cl₂ (2×200 mL). Theorganic layers were combined, washed sat. NaCl, dried MgSO₄, andchromatographed (silica, ethyl acetate) to afford the non-polydispersedtitle compound as a yellow oil (6.21 g, 71% yield).

Example 33 Mesylate of tetraethylene glycol monobenzylether (40)

[0446] To a solution of CH₂CI₂ (20 mL) was added non-polydispersedtetraethylene glycol monobenzylether 39 (6.21 g, 22 mmol) and cooled to0° C. in an ice bath. Then triethylamine (3.2 mL, 24 mmol) was added andthe reaction mixture was stirred for 15 min at 0° C. Thenmethanesulfonyl chloride (1.7 mL, 24 mmol) dissolved in CH₂CI₂ (2 mL)was added and the reaction mixture was stirred at 0° C. for 30 min, theice bath was removed and the reaction was stirred for an additional 2 hat room temperature. The crude reaction mixture was filtered throughCelite (washed CH₂CI₂, 80 mL) and the filtrate was washed H₂O (100 mL),5% NaHCO₃ (2×100 mL), H₂O (100 mL), sat. NaCl (100 mL), and dried MgSO₄.The resulting yellow oil was chromatographed on a pad of silicacontaining activated carbon (10 g) to afford the non-polydispersed titlecompound as a clear oil (7.10 g, 89% yield).

Example 34 Octaethylene glycol monobenzylether (41)

[0447] To a solution of tetrahydrofiran (140 mL) containing sodiumhydride (0.43 g, 18 mmol) was added dropwise a solution ofnon-polydispersed tetraethylene glycol (3.5 g, 18 mmol) intetrahydrofuran (10 mL) and the reaction mixture was stirred for 1 h.Then mesylate of non-polydispersed tetraethylene glycol monobenzylether40 (6.0 g, 16.5 mmol) dissolved in tetrahydrofuran (10 mL) was addeddropwise and the reaction mixture was stirred overnight. The crudereaction mixture was filtered through Celite (washed, CH₂Cl₂, 250 mL)and the filtrate was washed H₂O, dried MgSO₄, and evaporated to dryness.The resultant oil was chromatographed (silica, ethyl acetate/methanol,10:1) and chromatographed (silica, chloroform/methanol, 25:1) to affordthe non-polydispersed title compound as a clear oil (2.62 g, 34% yield).

Example 35 Synthesis of Stearate PEG8-Benzyl (43)

[0448] To a stirred cooled solution of non-polydispersed octaethyleneglycol monobenzylether 41 (0.998 g, 2.07 mmol) and pyridine (163.9 mg,2.07 mmol) was added non-polydispersed stearoyl chloride 42 (627.7 mg,2.07 mmol) in benzene. The reaction mixture was stirred overnight (18hours). The next day the reaction mixture was washed with water, driedover MgSO₄, concentrated and dried via vacuum. Then the crude productwas chromatographed on flash silica gel column, using 10% methanol/90%chloroform. The fractions containing the product were combined,concentrated and dried via vacuum to afford the non-polydispersed titlecompound.

Example 36 Hydrogenolysis of Stearate-PEG8-Benzyl

[0449] To a methanol solution of non-polydispersed stearate-PEG8-Bzl 43(0.854 g 1.138 mmol) Pd/C(10%) (palladium, 10% wt. on activated carbon)was added. The reaction mixture was stirred overnight (18 hours) underhydrogen. Then the solution was filtered, concentrated and purified byflash column chromatography using 10% methanol/90% chloroform, fractionswith R_(t)=0.6 collected, concentrated and dried to provide thenon-polydispersed acid 44.

Example 37 Activation of C18(PEG8) Oligomer

[0450] Two step activation of non-polydispersed stearate-PEG8 oligomerwas performed as described for stearate-PEG6 in Example 31 above toprovide the non-polydispersed activated C18(PEG8) oligomer 45.

Example 38 Synthesis of Activated Triethylene Glycol MonomethylOligomers

[0451] The following description refers to the scheme illustrated inFIG. 8. A solution of toluene containing 20% phosgene (100 ml,approximately 18.7 g, 189 mmol phosgene) was chilled to 0° C. under a N₂atmosphere. Non-polydispersed mTEG (triethylene glycol, monomethylether, 7.8 g, 47.5 mmol) was dissolved in 25 mL anhydrous ethyl acetateand added to the chilled phosgene solution. The mixture was stirred forone hour at 0° C., then allowed to warm to room temperature and stirredfor another two and one half hours. The remaining phosgene, ethylacetate and toluene were removed via vacuum distillation to leave thenon-polydispersed mTEG chloroformate 46 as a clear oily residue.

[0452] The non-polydispersed residue 46 was dissolved in 50 mL of drydichloromethane to which was added TEA (triethyleamine, 6.62 mL, 47.5mmol) and NHS (N-hydroxysuccinimide, 5.8 g, 50.4 mmol). The mixture wasstirred at room temperature under a dry atmosphere for twenty hoursduring which time a large amount of white precipitate appeared. Themixture was filtered to remove this precipitate and concentrated invacuo. The resultant oil 47 was taken up in dichloromethane and washedtwice with cold deionized water, twice with 1N HCl and once with brine.The organics were dried over MgSO₄, filtered and concentrated to providethe non-polydispersed title compound as a clear, light yellow oil. Ifnecessary, the NHS ester could be further purified by flashchromatography on silica gel using EtOAc as the elutant.

Example 39 Synthesis of Activated Palmitate-TEG Oligomers

[0453] The following description refers to the scheme illustrated inFIG. 9. Non-polydispersed palmitic anhydride (5 g; 10 mmol) wasdissolved in dry THF (20 mL) and stirred at room temperature. To thestirring solution, 3 mol excess of pyridine was added followed bynon-polydispersed triethylene glycol (1.4 mL). The reaction mixture wasstirred for 1 hour (progress of the reaction was monitored by TLC; ethylacetate-chloroform; 3:7). At the end of the reaction, THF was removedand the product was mixed with 10% H₂SO₄ acid and extracted ethylacetate (3×30 mL). The combined extract was washed sequentially withwater, brine, dried over MgSO₄, and evaporated to give non-polydispersedproduct 48. A solution of N,N′-disuccinimidyl carbonate (3 mmol) in DMF(˜10 mL) is added to a solution of the non-polydispersed product 48 (1mmol) in 10 mL of anydrous DMF while stirring. Sodium hydride (3 mmol)is added slowly to the reaction mixture. The reaction mixture is stirredfor several hours (e.g., 5 hours). Diethyl ether is added to precipitatethe activated oligomer. This process is repeated 3 times and the productis finally dried.

Example 40 Synthesis of Activated Hexaethylene Glycol MonomethylOligomers

[0454] The following description refers to the scheme illustrated inFIG. 10. Non-polydispersed activated hexaethylene glycol monomethylether was prepared analogously to that of non-polydispersed triethyleneglycol in Example 39 above. A 20% phosgene in toluene solution (35 mL,6.66 g, 67.4 mmol phosgene) was chilled under a N₂ atmosphere in anice/salt water bath. Non-polydispersed hexaethylene glycol 50 (1.85 mL,2.0 g, 6.74 mmol) was dissolved in 5 mL anhydrous EtOAc and added to thephosgene solution via syringe. The reaction mixture was kept stirring inthe ice bath for one hour, removed and stirred a further 2.5 hours atroom temperature. The phosgene, EtOAc, and toluene were removed byvacuum distillation, leaving non-polydispersed compound 51 as a clear,oily residue.

[0455] The non-polydispersed residue 51 was dissolved in 20 mL drydichloromethane and placed under a dry, inert atmosphere. Triethylamine(0.94 mL, 0.68 g, 6.7 mmol) and then NHS (N-hydroxy succinimide, 0.82 g,7.1 mmol) were added, and the reaction mixture was stirred at roomtemperature for 18 hours. The mixture was filtered through silica gel toremove the white precipitate and concentrated in vacuo. The residue wastaken up in dichloromethane and washed twice with cold water, twice with1 N HCl and once with brine. The organics were dried over Na₂SO₄,filtered and concentrated. Final purification was done via flashchromatography (silica gel, EtOAc) to obtain the UV activenon-polydispersed NHS ester 52.

Example 41 Synthesis of Polypeptide-Oligomer Conjugates

[0456] Mixtures of polypeptide-oligomer conjugates according to thepresent invention are synthesized as follows. A mixture of thepolypeptide is dissolved in anhydrous DMF. Then TEA and a mixture of anactivated oligomer of Example 18, 24, 29, 31, 37, 38, 39 or 40 inanhydrous THF is added. The reaction mixture is then stirred, preferablyfor 1 hour. The reaction mixture is acidified (for example, by adding 2mL of 0.1% TFA in water). The reaction is followed by HPLC. The reactionmixture is concentrated and purified by preparative liquidchromatography (for example, using Waters PrepLC™ 4000 RP Vydac C18Protein and peptide, 1×25 column, water/acetonitrile with 0.1% TFA,detection at 280 nm). Peaks corresponding to mono- or multi-conjugatedcompounds are isolated. Samples may be analyzed by MALDI-MS.

Example 42

[0457] The procedure of Example 41 is performed and the polypeptide isan adrenocorticotropic hormone peptide.

Example 43

[0458] The procedure of Example 41 is performed and the polypeptide isan adrenomedullin peptide.

Example 44

[0459] The procedure of Example 41 is performed and the polypeptide isan allatostatin peptide.

Example 45

[0460] The procedure of Example 41 is performed and the polypeptide isan amylin peptide.

Example 46

[0461] The procedure of Example 41 is performed and the polypeptide isan amyloid beta-protein fragment peptide.

Example 47

[0462] The procedure of Example 41 is performed and the polypeptide isan angiotensin peptide.

Example 48

[0463] The procedure of Example 41 is performed and the polypeptide isan antibiotic peptide.

Example 49

[0464] The procedure of Example 41 is performed and the polypeptide isan antigenic polypeptide.

Example 50

[0465] The procedure of Example 41 is performed and the polypeptide isan anti-microbial peptide.

Example 51

[0466] The procedure of Example 41 is performed and the polypeptide isan apoptosis related peptide.

Example 52

[0467] The procedure of Example 41 is performed and the polypeptide isan atrial natriuretic peptide.

Example 53

[0468] The procedure of Example 41 is performed and the polypeptide is abag cell peptide.

Example 54

[0469] The procedure of Example 41 is performed and the polypeptide is abombesin peptide.

Example 55

[0470] The procedure of Example 41 is performed and the polypeptide is abone GLA peptide.

Example 56

[0471] The procedure of Example 41 is performed and the polypeptide is abradykynin peptide.

Example 57

[0472] The procedure of Example 41 is performed and the polypeptide is abrain natriuretic peptide.

Example 58

[0473] The procedure of Example 41 is performed and the polypeptide is aC-peptide.

Example 59

[0474] The procedure of Example 41 is performed and the polypeptide is aC-type natriuretic peptide.

Example 60

[0475] 150 mg of salmon calcitonin (MW 3432, 0.043 mmol) was dissolvedin 30 ml of anhydrous DMF. Then TEA (35 μL) and the activated oligomerof Example 24 (42 mg, 0.067 mmol) in anhydrous THF (2 mL) was added. Thereaction was stirred for 1 hour, then acidified with 2 mL of 0.1% TFA inwater. The reaction was followed by HPLC. Then the reaction mixture wasconcentrated and purified by preparative liquid chromatography (WatersPrepLC™ 4000 RC Vydac C18 Protein and peptide, 1×25 column,water/acetonitrile with 0.1% TFA, detection at 280 nm). Two peaks,corresponding to mono- and di-conjugate were isolated. Samples wereanalyzed by MALDI-MS. MS for PEG7-octyl-sCT, mono-conjugate: 3897. MSfor PEG7-octyl-sCT, di-conjugate: 4361.

[0476] A similar procedure was used to conjugate salmon calcitonin withthe activated oligomer of Example 29. MS for PEG7-decyl-sCT,mono-conjugate: 3926. MS for PEG7-decyl-sCT, di-conjugate: 4420.

[0477] A similar procedure was used to conjugate salmon calcitonin withthe activated oligomer of Example 31. MS for stearate-PEG6-sCT,mono-conjugate: 4006. MS for stearate-PEG6-sCT, di-conjugate: 4582.

[0478] A similar procedure was used to conjugate salmon calcitonin withthe activated oligomer of Example 37. MS for stearate-PEG8-sCT,mono-conjugate: 4095.

[0479] A similar procedure is used to conjugate salmon calcitonin withthe activated oligomer of Example 18, 38, 39 and 40.

Example 61

[0480] The procedure of Example 41 is performed and the polypeptide is acalcitonin gene related peptide.

Example 62

[0481] The procedure of Example 41 is performed and the polypeptide is aCART peptide.

Example 63

[0482] The procedure of Example 41 is performed and the polypeptide is acasomorphin peptide.

Example 64

[0483] The procedure of Example 41 is performed and the polypeptide is achemotactic peptide.

Example 65

[0484] The procedure of Example 41 is performed and the polypeptide is acholecystokinin peptide.

Example 66

[0485] The procedure of Example 41 is performed and the polypeptide is acorticortropin releasing factor peptide.

Example 67

[0486] The procedure of Example 41 is performed and the polypeptide is acortistatin peptide.

Example 68

[0487] The procedure of Example 41 is performed and the polypeptide is adermorphin peptide.

Example 69

[0488] The procedure of Example 41 is performed and the polypeptide is adynorphin peptide.

Example 70

[0489] The procedure of Example 41 is performed and the polypeptide isan endorphin peptide.

Example 71

[0490] The procedure of Example 41 is performed and the polypeptide isan endothelin peptide.

Example 72

[0491] The procedure of Example 41 is performed and the polypeptide isan ETa receptor antagonist peptide.

Example 73

[0492] The procedure of Example 41 is performed and the polypeptide isan ETh receptor antagonist peptide.

Example 74

[0493] The procedure of Example 41 is performed and the polypeptide isan enkephalin peptide.

Example 75

[0494] The procedure of Example 41 is performed and the polypeptide is afibronectin peptide.

Example 76

[0495] The procedure of Example 41 is performed and the polypeptide is agalanin peptide.

Example 77

[0496] The procedure of Example 41 is performed and the polypeptide is agastrin peptide.

Example 78

[0497] The procedure of Example 41 is performed and the polypeptide is aglucagon peptide.

Example 79

[0498] The procedure of Example 41 is performed and the polypeptide is aGn-RH associated peptide.

Example 80

[0499] The procedure of Example 41 is performed and the polypeptide is agrowth factor peptide.

Example 81

[0500] Human growth hormone was conjugated with the activated oligomersof Example 40 as illustrated in FIG. 10. Human growth hormone(somatropin (rDNA origin) for injection), available under the trade nameSaizen™ from Serono of Randolph, Mass., was dissolved in DMSO such thatthe hGH was at a 0.58 mmol concentration. TEA (278 equivalents) wasadded and the solution was stirred for approximately ten minutes. Twoequivalents, five equivalents or thirty equivalents of activatedhexaethylene glycol 52 was added from a 0.2 M solution of the activatedoligomer in dry THF. Reactions were stirred at room temperature for 45minutes to one hour. Aliquots of each reaction mixture were quenched in600 μL of 0.1% TFA in water. HPLC comparison of the 2 polymer equivalentand the 5 polymer equivalent reaction mixtures vs. unconjugated hGH isshown in FIG. 14. HPLC analysis of the thirty polymer equivalentreaction is shown in FIG. 15.

[0501] Samples of the conjugates for mass spectroscopy were purified viaanalytical HPLC using a reversed-phase C₁₈ column and awater/acetonitrile gradient. The entire peak from the 2 equivalentreaction mixture was collected, concentrated and analyzed using MALDImass spectroscopy. The mass spectra of this material showed evidence ofthe presence of mono-conjugated, di-conjugated, tri-conjugated andtetra-conjugated hGH as well as some remaining unreacted hGH (FIG. 16).The five equivalent reaction mixture was purified crudely according topolarity as indicated in FIG. 17. MALDI mass spectra of the concentratedfractions (FIG. 18, FIG. 19 and FIG. 20) indicated that the level ofconjugation of the protein increased with retention time. Electrospraymass spectra of fraction E, FIG. 21, gave results consistent with thepresence of hexa-conjugated hGH. The entire peak from the thirty polymerequivalent reaction mixture was collected and concentrated. Electrospraymass spectral analysis, FIG. 22, showed deca- and higher conjugatedmaterial.

[0502] A similar procedure is used to conjugate hGH with the activatedoligomer of Example 18, 24, 29, 31 or 37.

Example 82 Synthesis of Human Growth Hormone-Oligomer Conjugates withActivated Palmitate-TEG Oligomers

[0503] Procedures similar to those described above in Example 81 wereperformed using the activated polymer from Example 39. Progress ofconjugation was checked by HPLC by taking 20 μL of the conjugatedreaction mixture in a vial and diluting with 100 μL of 0.1%TFA-water-IPA (1:1), the results of which are illustrated in FIG. 23.After 2 hours, the reaction was quenched by adding 0.1% TFA-water. Theconjugated product was purified by prep. HPLC.

Example 83 Synthesis of Human Growth Hormone-Oligomer Conjugates withActivated TEG Oligomers

[0504] One equivalent of human growth hormone (hGH) (somatropin (rDNAorigin) for injection), available under the trade name Saizen™ fromSerono of Randolph, Mass. was dissolved in DMSO (1 mg/125 μL) andstirred at room temperature for 2-4 minutes. Two equivalents TEA wasadded followed by two equivalents of the activated oligomer of Example38, which was dissolved in THF. After 2 hours, the reaction was quenchedby adding 0.1% TFA-water. The conjugated product was purified by prep.HPLC as illustrated in FIG. 24.

[0505] A similar procedure five equivalents TEA and five equivalents ofthe activated oligomer of Example 39 was performed. The conjugatedproduct was purified by prep. HPLC using C18 column as illustrated inFIG. 25. The mobile phase and elution time were as follows: Time mL/minSolvent A Solvent B  0 3.5 80  20 55 3.5  0 100

[0506] The pooled fraction was lyophilized into a white powder. The massspectra for the compound are illustrated in FIGS. 26 and 27.

[0507] A similar procedure utilizing nine equivalents TEA and nineequivalents of the activated oligomer of Example 39 was performed. Theconjugated product was purified by prep. HPLC using C18 column asillustrated in FIG. 28.

Example 84

[0508] The procedure of Example 41 is performed and the polypeptide is aGTP-binding peptide.

Example 85

[0509] The procedure of Example 41 is performed and the polypeptide is aguanylin peptide.

Example 86

[0510] The procedure of Example 41 is performed and the polypeptide isan inhibin peptide.

Example 87 Synthesis of Insulin-Oligomer Conjugates

[0511] To human insulin (zinc or zinc free, 2g, 0.344 mmol based on dryweight) in 25 mL dimethylsulfoxide (>99% purity) at 22±4° C. was added 8mL triethyl amine (>99% purity). The resulting mixture was stirred for 5to 10 minutes at 22±4° C. To the above was rapidly added the activatedoligomer of Example 18 above (0.188 g, 0.36 mmol based on 100%activation) in 7.5 mL acetonitrile under stirring at 22±4° C. Thesolution was stirred for 45 minutes and quenched with acetic acidsolution with maintaining the temperature below 27° C. The reaction wasmonitored by analytical HPLC. This reaction condition producesPEG7-hexyl-insulin, monoconjugated at the B29 position(PEG7-hexyl-insulin, B29 monoconjugated) at yield 40-60%. The crudereaction mixture (PEG7-hexyl-insulin, B29 monoconjugated, 40-60%,unreacted insulin 8-25%, related substances 15-35%) was dialyzed ordifiltered (3000-3500 molecular weight cut off, MWCO) to remove organicsolvents and small molecular weight impurities, exchanged againstammonium acetate buffer and lyophilized.

[0512] The conjugation reaction of PEG7-hexyl-insulin, monoconjugated atthe B29 position, was monitored by analytical HPLC. This analytical HPLCmethod used a Waters Delta-Pak C18 column, 150×3.9 mm I.D., 5 μm, 300 Å.The solvent system consisted of Solvent B: 0.1% TFA in 50/50methanol/water, and Solvent D: 0.1% TFA in methanol. The gradient systemwas as follows: Flow rate Time (min) % Solvent B % Solvent D (mL/min)Initial (0) 100 0 1.00 20  40 60  1.00 25 100 0 1.00

[0513] A similar procedure is used to provide non-polydispersed mixturesof insulin-oligomer conjugates using the activated oligomers of Example24, 29, 31, 37, 38, 39 and 40.

Example 88

[0514] The procedure of Example 41 is performed and the polypeptide isan interleukin peptide.

Example 89

[0515] The procedure of Example 41 is performed and the polypeptide is aleptin peptide.

Example 90

[0516] The procedure of Example 41 is performed and the polypeptide is aleucokinin peptide.

Example 91

[0517] The procedure of Example 41 is performed and the polypeptide is aluteinizing hormone-releasing hormone.

Example 92

[0518] The procedure of Example 41 is performed and the polypeptide is amastoparan peptide.

Example 93

[0519] The procedure of Example 41 is performed and the polypeptide is amast cell degranulating peptide.

Example 94

[0520] The procedure of Example 41 is performed and the polypeptide is amelanocyte stimulating hormone peptide.

Example 95

[0521] The procedure of Example 41 is performed and the polypeptide is amorphiceptin peptide.

Example 96

[0522] The procedure of Example 41 is performed and the polypeptide is amotilin peptide.

Example 97

[0523] The procedure of Example 41 is performed and the polypeptide is aneuro-peptide.

Example 98

[0524] The procedure of Example 41 is performed and the polypeptide is aneuropeptide Y peptide.

Example 99

[0525] The procedure of Example 41 is performed and the polypeptide is aneurotropic factor peptide.

Example 100

[0526] The procedure of Example 41 is performed and the polypeptide isan orexin peptide.

Example 101

[0527] The procedure of Example 41 is performed and the polypeptide isan opioid peptide.

Example 102

[0528] The procedure of Example 41 is performed and the polypeptide isan oxytocin peptide.

Example 103

[0529] The procedure of Example 41 is performed and the polypeptide is aPACAP peptide.

Example 104

[0530] The procedure of Example 41 is performed and the polypeptide is apacreastatin peptide.

Example 105

[0531] The procedure of Example 41 is performed and the polypeptide is apancreatic polypeptide.

Example 106

[0532] The procedure of Example 41 is performed and the polypeptide is aparathyroid hormone peptide.

Example 107

[0533] The procedure of Example 41 is performed and the polypeptide is aparathyroid hormone-related peptide.

Example 108

[0534] The procedure of Example 41 is performed and the polypeptide is apeptide T peptide.

Example 109

[0535] The procedure of Example 41 is performed and the polypeptide is aprolactin-releasing peptide.

Example 110

[0536] The procedure of Example 41 is performed and the polypeptide is apeptide YY peptide.

Example 111

[0537] The procedure of Example 41 is performed and the polypeptide is arenin substrate peptide.

Example 112

[0538] The procedure of Example 41 is performed and the polypeptide is asecretin peptide.

Example 113

[0539] The procedure of Example 41 is performed and the polypeptide is asomatostatin peptide.

Example 114

[0540] The procedure of Example 41 is performed and the polypeptide is asubstance P peptide.

Example 115

[0541] The procedure of Example 41 is performed and the polypeptide is atachykinin peptide.

Example 116

[0542] The procedure of Example 41 is performed and the polypeptide is athyrotropin-releasing hormone peptide.

Example 117

[0543] The procedure of Example 41 is performed and the polypeptide is atoxin peptide.

Example 118

[0544] The procedure of Example 41 is performed and the polypeptide is avasoactive intestinal peptide.

Example 119

[0545] The procedure of Example 41 is performed and the polypeptide is avasopressin peptide.

Example 120

[0546] The procedure of Example 41 is performed and the polypeptide is avirus related peptide.

Example 121 Purification of B29 Modified PEG7-Hexyl-Insulin,Monoconjugate, from the Crude Mixture

[0547] PEG7-hexyl-insulin, B29 monoconjugated, was purified from thecrude mixture of Example 87 using a preperative HPLC system. Lyophilizedcrude mixture (0.5 g, composition: PEG7-hexyl-insulin, B29monoconjugated, 40-60%, unreacted insulin 8-25%, related substances15-35%) was dissolved in 5-10 mL 0.01 M ammonium acetate buffer, pH 7.4and loaded to a C-18 reverse phase HPLC column (150×3.9 mm) equilibratedwith 0.5% triethylamine/0.5%/phosphoric acid buffer TEAP A). The columnwas eluted with a gradient flow using TEAP A and TEAP B (80%acetonitrile and 20% TEAP A) solvent system. The gradient system forpreparative HPLC purification of PEG7-hexyl-insulin, B29 monoconjugate,from the crude mixture was as follows: Time Flow rate (min) % TEAP A %TEAP B (mL/min) Initial (0) 70 30 30  45 64 36 30 105 60 40 30 115 40 6030 125 15 85 30 135 15 85 30

[0548] Fractions were analyzed by HPLC and the product fractions thatwere >97% purity of PEG7-hexyl-insulin, B29 monoconjugate, were pooled.The elution buffer and solvent were removed by dialysis or diafiltration(MWCO 3000-3500) against ammonium acetate buffer (0.01M, pH 7.4)andexchanged into ammonium acetate buffer and lyophilized to produce whitepowder of PEG7-hexyl-insulin, B29 monoconjugate (purity >97%).

[0549] An analytical HPLC method using the same column and solventsystem as the method used in Example 87 to monitor the reaction was usedfor analysis of PEG7-hexyl-insulin, B29 monoconjugate. However, thegradient conditions were as follows: Flow rate Time (min) % Solvent B %Solvent D (mL/min) Initial (0) 100 0 1.00 30  10 90  1.00 35 100 0 1.00

Example 122 Determination of the Dispersity Coefficient for a Mixture ofHuman Insulin-Oligomer Conjugates

[0550] The dispersity coefficient of a mixture of human insulin-oligomerconjugates is determined as follows. A mixture of human insulin-oligomerconjugates is provided, for example as described above in Example 87. Afirst sample of the mixture is purified via HPLC to separate and isolatethe various human insulin-oligomer conjugates in the sample. Assumingthat each isolated fraction contains a purely monodispersed mixture ofconjugates, “n” is equal to the number of fractions collected. Themixture may include one or more of the following conjugates, which aredescribed by stating the conjugation position followed by the degree ofconjugation: Gly^(A1) monoconjugate; Phe^(B1) monoconjugate; Lys^(B29)monoconjugate; Gly^(A1), Phe^(B1) diconjugate; Gly^(A1), Lys^(B29)diconjugate; Phe^(B1), Lys^(B29) diconjugate; and/or Gly^(A1), Phe^(B1),Lys^(B29) triconjugate. Each isolated fraction of the mixture isanalyzed via mass spectroscopy to determine the mass of the fraction,which allows each isolated fraction to be categorized as a mono-, di-,or tri-conjugate and provides a value for the variable “M_(i)” for eachconjugate in the sample.

[0551] A second sample of the mixture is analyzed via HPLC to provide anHPLC trace. Assuming that the molar absorptivity does not change as aresult of the conjugation, the weight percent of a particular conjugatein the mixture is provided by the area under the peak of the HPLC tracecorresponding to the particular conjugate as a percentage of the totalarea under all peaks of the HPLC trace. The sample is collected andlyophilized to dryness to determine the anhydrous gram weight of thesample. The gram weight of the sample is multiplied by the weightpercent of each component in the sample to determine the gram weight ofeach conjugate in the sample. The variable “N_(i)” is determined for aparticular conjugate (the i^(th) conjugate) by dividing the gram weightof the particular conjugate in the sample by the mass of the particularconjugate and multiplying the quotient by Avagadro's number(6.02205×10²³ mole⁻¹), M₁, determined above, to give the number ofmolecules of the particular conjugate, N₁, in the sample. The dispersitycoefficient is then calculated using n, M_(i) as determined for eachconjugate, and N_(i) as determined for each conjugate.

Example 123 Cytosensor Studies for Insulin-Oligomer Conjugates

[0552] Colo 205 (colorectal adenocarcinoma cells from ATCC, catalog#CCL-222) cells that had been serum-deprived for approximately 18 hourswere suspended in 3:1 Cytosensor low-buffer RPMI-1640 media: Cytosensoragarose entrapment media and seeded into Cytosensor capsule cups at100,000 cells/10 μL droplet. Cells were allowed to equilibrate on theCytosensor to the low-buffer RPMI-1640 media at a flow rate of 100 μLper minute for approximately 3 hours until baseline acidification rateswere stable. Insulin drugs (insulin or insulin conjugates) were dilutedto 50 nM in low-buffer RPMI-1640 media and applied to the cells for 20minutes at 100 μL/minute. Following the exposure, the drug solutionswere withdrawn and the cells were again perfused under the continuousflow of low-buffer media alone. Data collection continued untilacidification rates returned to baseline levels (approximately one hourfrom application of drug solutions). The results are illustrated in FIG.29. As used in FIG. 29, insulin is human insulin; PEG4 is anon-polydispersed mixture of mPEG4-hexy-insulin, B29 monoconjugates;PEG10 is a non-polydispersed mixture of mPEG10-hexyl-insulin, B29monoconjugates; PEG7 is a non-polydispersed mixture ofmPEG7-hexyl-insulin, B29 monoconjugates; PEG7_(AVG) is a polydispersedmixture of mPEG7_(AVG)-hexyl-insulin, B29 monoconjugates.

Example 124 Enzymatic Stability of Insulin-Oligomer Conjugates

[0553] Chymotryp sin digests were conducted in a phosphate buffer, pH7.4, at 37° C. in a shaking water bath. The insulin/insulin conjugateconcentration was 0.3 mg/mL. The chymotrypsin concentration was2Units/mL. 100 μL samples were removed at the indicated time points andquenched with 25 μL of a 1:1 mixture of 0.1% trifluoroaceticacid:isopropyl alcohol. Samples were analyzed by reverse phase HPLC andthe relative concentrations of insulin/insulin conjugate were determinedby calculating the areas under the curves.

[0554] As used in FIG. 30, insulin is human insulin; PEG4 is anon-polydispersed mixture of mPEG4-hexyl-insulin, B29 monoconjugates;PEG10 is a non-polydispersed mixture of mPEG10-hexyl-insulin, B29monoconjugates; PEG7 is a non-polydispersed mixture ofmPEG7-hexyl-insulin, B29 monoconjugates; PEG7_(AVG) is a polydispersedmixture of mPEG7_(AVG)-hexyl-insulin, B29 monoconjugates.

Example 125 Dose Dependent Activity for Insulin-Oligomer Conjugates

[0555] An effective animal model for evaluating formulations uses normalfasted beagle dogs. These dogs are given from 0.25 mg/kg to 1.0 mg/kg ofinsulin conjugates to evaluate the efficacy of various formulations.This model was used to demonstrate that insulin conjugates according tothe present invention provide lower glucose levels in a dose dependentmanner better than polydispersed insulin conjugates, which are not partof the present invention and are provided for comparison purposes.

[0556] The protocol for dog experiments calls for a blood glucosemeasurement at time zero just before a drug is administered. Theformulation in solid oral dosage form is then inserted into the dog'smouth. Blood is drawn at 15, 30, 60 and 120 minutes and glucose levelsare measured and graphed. The lower the glucose levels, the better theactivity of the insulin conjugate. In FIG. 31, the glucose lowering, andthus the activity, of the conjugates of the present invention is shownto be dose dependent. For comparison purposes, FIG. 32 shows that theglucose lowering of polydispersed insulin conjugates in a capsuleformulation, which are not a part of the present invention, is less dosedependent than conjugates of the present invention.

Example 126 Activity and Inter-Subject Variability for Insulin-OligomerConjugates

[0557] An effective animal model for evaluating formulations uses normalfasted beagle dogs. These dogs are given 0.25 mg/kg of insulinconjugates to evaluate the efficacy of various formulations. This modelwas used to demonstrate that insulin conjugates according to the presentinvention provide lower inter-subject variability and better activitythan polydispersed insulin conjugates, which are not part of the presentinvention but are provided for comparison purposes.

[0558] The protocol for dog experiments calls for a blood glucosemeasurement at time zero just before a drug is administered. The oralliquid dosage formulation is then squirted into the back of the dog'smouth. In each case, the dogs received 0.25 mg/kg of this solution.Blood is drawn at 15, 30, 60 and 120 minutes and glucose levels aremeasured and graphed. The lower the glucose levels, the better theactivity of the insulin conjugate. In FIGS. 33, 34 and 35, the resultsobtained with PEG4-hexyl-insulin, monoconjugate; PEG7-hexyl-insulin,monoconjugate; and PEG10-hexyl-insulin, monoconjugate, respectively,show these PEG conjugates of the present invention result in lessinter-subject variability and higher activity than the results shown inFIG. 36 for the polydispersed PEG7_(AVG)-hexyl-insulin, monoconjugate,which is not a part of the present invention and is provided forcomparison purposes.

Example 127 Cytosensor® Studies for Calcitonin-Oligomer Conjugates

[0559] T-47D cells (mammary ductal carcinoma cell line, obtained fromAmerican Type Culture Collection were suspended at a density of 1×10⁷cells/mL in running buffer (low-buffered, serum-free, bicarbonate-freeRPMI 1640 medium from Molecular Devices of Sunnyvale, Calif.Approximately 100,000 cells were then immobilized in an agarose cellentrapment medium in a 10 μL droplet and sandwiched between two 3-μmpolycarbonate membranes in a cytosensor capsule cup. Cytosensor capsulecups placed in sensor chambers on the Cytosensor® Microphysiometer werethen held in very close proximity to pH-sensitive detectors. Runningbuffer was then pumped across the cells at a rate of 100 μL/min exceptduring 30-second intervals when the flow was stopped, and acidificationof the running buffer in the sensor chamber was measured. Acidificationrates were determined every 2 minutes. The temperature of the sensorchambers was 37° C. Cells were allowed to equilibrate in the sensorchambers for 2-3 hours prior to the start of the experiment during whichtime basal acidification rates were monitored. Cells were then exposedto test compounds (Salmon Calcitonin or Octyl-Di-Calcitonin) diluted inrunning buffer at various nM concentration. Exposure of cells to testcompounds occurred for the first 40 seconds of each 2 minute pump cyclein a repeating pattern for a total of 20 minutes. This allowedsufficient exposure of the cells to the test compounds to elicit areceptor-mediated response in cellular metabolism followed byapproximately 50 seconds of flow of the running buffer containing nocompounds. This procedure rinsed away test solutions (which had aslightly lower pH than running buffer alone) from the sensor chamberbefore measuring the acidification rate. Thus, the acidification rateswere solely a measure of cellular activity. A similar procedure was usedto obtain data for PEG7-octyl-sCT, monoconjugate (Octyl-Mono);PEG7-decyl-sCT, monoconjugate (Decyl-Mono); PEG7-decyl-sCT, diconjugate(Decyl-Di); stearate-PEG6-sCT, monoconjugate (PEG6 St. Mono); andstearate-PEG8-sCT, monoconjugate (PEG8 St. Mono). Data was analyzed forrelative activity of compounds by calculating the Area Under the Curve(AUC) for each cytosensor chamber acidification rate graph and plottedas a bar chart illustrated in FIG. 37 showing average AUC measurementstaken from multiple experiments performed under the same experimentalconditions.

Example 128 Enzymatic Stability for Calcitonin-Oligomer Conjugates

[0560] Compounds, supplied as lyophilized powders, are resuspended in 10mM phosphate buffer pH 7.4 and then submitted for concentrationdetermination by HPLC. The phosphate buffer is used to create a solutionwith a pH that is optimum for activity of each particular gut enzyme.Aliquots of the compound thus prepared are transferred to 1.7 mLmicrocentrifuge tubes and shaken in a 37° C. water bath for 15 minutesto allow compounds to equilibrate to temperature. After 15 minutes, 2 μLof the appropriate concentrated gut enzyme is added to each tube toachieve the final concentration desired. Chymotrypsin and trypsin areresuspended in 1 mM HCl. Also, as a control, compounds are treated with2 μL of 1 mM HCl. Immediately following additions, 100 μL of sample isremoved from the control tube and quenched with either 25 μL ofchymotrypsin/trypsin quenching solution (1:1 1% TFA:Isopropanol). Thissample will serve as T=0 min. A sampling procedure is repeated atvarious time intervals depending on the gut enzyme used. Chymotrypsinhas 15, 30 and 60 minute samples. Trypsin has 30, 60, 120 and 180 minutesamples. Once all points have been acquired, a final sample is removedfrom the control tube to make sure that observed degradation is nottemperature or buffer related. The chymotrypsin and trypsin samples maybe collected directly into HPLC vials. RP-HPLC (acetonitrile gradient)is used to determine AUC for each sample and % degradation is calculatedbased from the T=0 min control. The results are provided below in Tables1 to 4. TABLE 1 % Remaining Following 0.5 U/mL Chymotrypsin Digest ofPEG7-Octyl-Salmon Calcitonin, Diconjugate Time Non-Formulated BufferedFormulation 15 63 71 68 69 88 86 88 30 34 48 50 46 73 88 86 60  6 15 2015 61 69 84 Control Control 60 104  88 97 103  116  104  101 

[0561] TABLE 2 % Remaining Following 0.5 U/mL Chymotrypsin Digest ofSalmon Calcitonin (for comparison purposes; not part of the invention)Time Non-Formulated 10 73 Buffered Formulation 15 — 55 62 35 66 59 91 9230 30 26 40 13 42 54 86 87 60 1.6  5 12  1 12 55 82 85 Control Control60 — 100  93 45 100  102  98 103 

[0562] TABLE 3 % Remaining following 1 U/mL Trypsin Digest ofPEG7-Octyl-Salmon Calcitonin, Diconjugate Time Non-Formulated  30 87 8983 90  60 78 86 76 85 120 72 82 68 78 180 — 81 61 73 Control  60 103 100  120 106  105  99 180 104  99

[0563] TABLE 4 % Remaining following 1 U/mL Trypsin Digest of SalmonCalcitonin (for comparison purposes; not part of the invention) TimeNon-Formulated  30 80 50 82 87  60 66 28 69 76 120 44  7 46 59 180 —  231 46 Control  60 41 101  120 69 16 102  180  7 101 

Example 130 Activity and Inter-Subject Variability forCalcitonin-Oligomer Conjugates

[0564] Male CF-1 mice (Charles River, Raleigh, N.C.) weighing 20-25 gwere housed in the Nobex vivarium in a light-(L:D cycle of 12:12, lightson at 0600 h), temperature-(21-23° C.), and humidity-(40-60% relativehumidity) controlled room. Animals were permitted free access tolaboratory chow (PMI Nutrition) and tap water. Mice were allowed toacclimate to housing conditions for 48-72 hours prior to the day ofexperiment.

[0565] Prior to dosing, mice were fasted overnight and water wasprovided ad libitum. Mice were randomly distributed into groups of fiveanimals per time point and were administered a single oral dose of aPEG7-octyl-sCT, diconjugate (Octyl Di) according to the presentinvention or salmon calcitonin (sCT or Calcitonin) for comparisonpurposes. Oral doses were administered using a gavaging needle (Popper#18, 5 cm from hub to bevel) at 10 mL/kg in the following 0.2 μg/mLphosphate-buffered PEG7-octyl-sCT, diconjugate, formulation: IngredientAmount PEG7-octyl-sCT, 20 μg diconjugate Sodium-cholate 2.5 gSodium-deoxy-cholate 2.5 g Sodium phosphate buffer, q.s. to 100 g 100mM, pH 7.4

[0566] The buffered formulation was prepared by adding 80 mL ofphosphate buffer in a clean tared glass beaker. The sodium cholate wasslowly added to the phosphate buffer with stirring until dissolved. Thedeoxy cholate was then added and stirring was continued until dissolved.The PEG7-octyl-sCT, diconjugate, solution equivalent to 20 μg was added.Finally, the remaining phosphate buffer was added to achieve a finalweight of 100 g. Vehicle-control mice were used in all experiments.Dose-response curves were constructed using a single time point 60minutes after drug administration. These curves are illustrated in FIGS.38-41.

[0567] At appropriate time points, mice were ether-anesthetized, thevena cavae exteriorized, and blood samples were obtained via a syringefitted with a 25-gauge needle. Blood aliquots were allowed to clot at22° C. for 1 hour, and the sera removed and pipetted into a cleanreceptacle. Total serum calcium was determined for each animal using acalibrated Vitros DT60 II analyzer.

[0568] Serum calcium data were plotted and pharmacokinetic parametersdetermined via curve-fitting techniques using SigmaPlot software(Version 4.1). Means and standard deviations (or standard errors) werecalculated and plotted to determine effect differences among dosinggroups. Average serum calcium data for various conjugates are providedin Table 5 below. TABLE 5 % Baseline Calcium Drop Conjugate Dispersityat 2.0 μg/kg dose PEG7-Octyl-sCT, diconjugate Monodispersed mixture 21.0Stearate-PEG6-sCT, diconjugate Monodispersed mixture 16.0PEG7-Decyl-sCT, monoconjugate Monodispersed mixture 11.5Stearate-PEG8-sCT, diconjugate Monodispersed mixture 11.0PEG7-Decyl-sCT, diconjugate Monodispersed mixture 8.3

[0569] Despite an in vitro activity as determined in Example 50 abovethat may not be comparable with the in vitro activity of PEG7-octyl-sCTand PEG7-decyl-sCT mono- and di-conjugates, the stearate-PEG6-sCT,diconjugate, and stearate-PEG8-sCT, diconjugate, appear to have in vivoactivity (as evidenced by the drops in % baseline calcium from Table 5above) that are comparable with the in vivo activity observed for thePEG7-octyl-sCT and PEG7-decyl-sCT, mono- and di-conjugates. While notwanting to be bound by a particular theory, the improved in vivoactivity of the stearate containing conjugates may indicate that theseconjugates are undergoing hydrolysis in vivo to provide an active salmoncalcitonin or active salmon calcitonin-PEG conjugate.

Example 131

[0570] The assay is as follows:

[0571] Cell culture: Stable clones expressing the full length human GHRwere generated in 293 cells (human kidney embryonal cell line),designated 293GHR, as previously described.

[0572] Transcription assays: These were performed in 293 GHR cellstransiently transfected with a reported construct containing aStat5-binding element (LHRE) fused to a minimal TK promoter andluciferase. A β-galactosidase expression vector was cotransfected as atransfection control and luciferase values corrected for β-galactosidaseactivity. Sixteen hours after transfection, cells were transferred intoserum free medium and treated with GH or agonist for 6 hours. Luciferaseactivity is reported as percentage of maximal activity stimulated by GHin the specific experiment to allow comparison between repeatedexperiments. The maximal activity stimulated by GH is the fold inductionstimulated by GH, i.e. corrected luciferase value in GH stimulated cellsdivided by corrected luciferase value in unstimulated cells. Results ofthe assay are shown in FIGS. 42 and 43 where Genotropin is human growthhormone (standard, not part of the present invention), GH-002 is a 2equivalent mTEG conjugate, GH-003 is a 5 equivalent mTEG conjugate,GH-004 is a 5 equivalent mTEG conjugate, Prot hGH is human growthhormone (standard, not part of the present invention), and hGH-TEG is a9 equivalent mTEG conjugate.

[0573] In the specification, there has been disclosed typical preferredembodiments of the invention and, although specific terms are employed,they are used in a generic and descriptive sense only and not forpurposes of limitation, the scope of the invention being set forth inthe following claims.

What is claimed is:
 1. A substantially monodispersed mixture ofconjugates, each conjugate comprising a drug coupled to an oligomer thatcomprises a polyalkylene glycol moiety.
 2. The mixture according toclaim 1, wherein the polyalkylene glycol moiety has at least 2, 3 or 4polyalkylene glycol subunits.
 3. The mixture according to claim 1,wherein the polyalkylene glycol moiety has at least 5 or 6 polyalkyleneglycol subunits.
 4. The mixture according to claim 1, wherein thepolyalkylene glycol moiety has at least 7 polyalkylene glycol subunits.5. The mixture according to claim 4, wherein the oligomer is covalentlycoupled to the drug.
 6. The mixture according to claim 4, wherein theoligomer further comprises a lipophilic moiety.
 7. The mixture accordingto claim 4, wherein the polyalkylene glycol moiety is a lower alkylpolyalkylene glycol moiety.
 8. The mixture according to claim 7, whereinthe lower alkyl polyalkylene glycol moiety is a polyethylene glycolmoiety.
 9. The mixture according to claim 8, wherein the oligomerfurther comprises a lipophilic moiety.
 10. The mixture according toclaim 7, wherein the lower alkyl polyalkylene glycol moiety is apolypropylene glycol moiety.
 11. The mixture according to claim 10,wherein the polypropylene glycol moiety is uniform.
 12. The mixtureaccording to claim 11, wherein the oligomer is devoid of a lipophilicmoiety, and wherein the conjugate is amphiphilically balanced such thatit is aqueously soluble and able to penetrate biological membranes. 13.The mixture according to claim 1, wherein at least 96, 97, 98 or 99percent of the conjugates in the mixture have the same molecular weight.14. The mixture according to claim 1, wherein the mixture is amonodispersed mixture.
 15. The mixture according to claim 1, wherein themixture is a substantially purely monodispersed mixture.
 16. The mixtureaccording to claim 1, wherein at least 96, 97, 98 or 99 percent of theconjugates in the mixture have the same molecular weight and the samemolecular structure.
 17. The mixture according to claim 1, wherein themixture is a purely monodispersed mixture.
 18. The mixture according toclaim 17, wherein the oligomer is covalently coupled to the drug. 19.The mixture according to claim 17, wherein the oligomer furthercomprises a lipophilic moiety.
 20. The mixture according to claim 17,wherein the polyalkylene glycol moiety is a lower alkyl polyalkyleneglycol moiety.
 21. The mixture according to claim 20, wherein the loweralkyl polyalkylene glycol moiety is a polyethylene glycol moiety. 22.The mixture according to claim 21, wherein the oligomer furthercomprises a lipophilic moiety.
 23. The mixture according to claim 20,wherein the lower alkyl polyalkylene glycol moiety is a polypropyleneglycol moiety.
 24. The mixture according to claim 23, wherein thepolypropylene glycol moiety is uniform.
 25. The mixture according toclaim 24, wherein the oligomer is devoid of a lipophilic moiety, andwherein the conjugate is amphiphilically balanced such that it isaqueously soluble and able to penetrate biological membranes.
 26. Themixture according to claim 1, wherein the mixture has an in vivoactivity that is greater than the in vivo activity of a polydispersedmixture of drug-oligomer conjugates having the same number averagemolecular weight as the mixture.
 27. The mixture according to claim 1,wherein the mixture has an in vitro activity that is greater than the invitro activity of a polydispersed mixture of drug-oligomer conjugateshaving the same number average molecular weight as the mixture.
 28. Themixture according to claim 1, wherein the mixture has an increasedresistance to degradation by chymotrypsin when compared to theresistance to degradation by chymotrypsin of a polydispersed mixture ofdrug-oligomer conjugates having the same number average molecular weightas the mixture.
 29. The mixture according to claim 1, wherein themixture has an inter-subject variability that is less than theinter-subject variability of a polydispersed mixture of drug-oligomerconjugates having the same number average molecular weight as themixture.
 30. The mixture according to claim 1, wherein the drug is apolypeptide.
 31. The mixture according to claim 30, wherein thepolypeptide is selected from the group consisting of adrenocorticotropichormone peptides, adrenomedullin peptides, allatostatin peptides, amylinpeptides, amyloid beta-protein fragment peptides, angiotensin peptides,antibiotic peptides, antigenic polypeptides, anti-microbial peptides,apoptosis related peptides, atrial natriuretic peptides, bag cellpeptides, bombesin peptides, bone GLA peptides, bradykinin peptides,brain natriuretic peptides, C-peptides, C-type natriuretic peptides,calcitonin peptides, calcitonin gene related peptides, CART peptides,casomorphin peptides, chemotactic peptides, cholecystokinin peptides,colony-stimulating factor peptides, corticortropin releasing factorpeptides, cortistatin peptides, cytokine peptides, dermorphin peptides,dynorphin peptides, endorphin peptides, endothelin peptides, ETareceptor antagonist peptides, ETh receptor antagonist peptides,enkephalin peptides, fibronectin peptides, galanin peptides, gastrinpeptides, glucagon peptides, Gn-RH associated peptides, growth factorpeptides, growth hormone peptides, GTP-binding protein fragmentpeptides, guanylin peptides, inhibin peptides, insulin peptides,interleukin peptides, laminin peptides, leptin peptides, leucokininpeptides, luteinizing hormone-releasing hormone peptides, mastoparanpeptides, mast cell degranulating peptides, melanocyte stimulatinghormone peptides, morphiceptin peptides, motilin peptides,neuro-peptides, neuropeptide Y peptides, neurotropic factor peptides,orexin peptides, opioid peptides, oxytocin peptides, PACAP peptides,pancreastatin peptides, pancreatic polypeptides, parathyroid hormonepeptides, parathyroid hormone-related peptides, peptide T peptides,prolactin-releasing peptides, peptide YY peptides, renin substratepeptides, secretin peptides, somatostatin peptides, substance Ppeptides, tachykinin peptides, thyrotropin-releasing hormone peptides,toxin peptides, vasoactive intestinal peptides, vasopressin peptides,and virus related peptides.
 32. The mixture according to claim 31,wherein the oligomer is covalently coupled to a nucleophilic residue ofthe polypeptide.
 33. The mixture according to claim 31, wherein theoligomer further comprises a lipophilic moiety.
 34. The mixtureaccording to claim 31, wherein the polyalkylene glycol moiety is a loweralkyl polyalkylene glycol moiety.
 35. The mixture according to claim 34,wherein the lower alkyl polyalkylene glycol moiety is a polyethyleneglycol moiety.
 36. The mixture according to claim 35, wherein theoligomer further comprises a lipophilic moiety.
 37. The mixtureaccording to claim 34, wherein the lower alkyl polyalkylene glycolmoiety is a polypropylene glycol moiety.
 38. The mixture according toclaim 37, wherein the polypropylene glycol moiety is uniform.
 39. Themixture according to claim 38, wherein the oligomer is devoid of alipophilic moiety, and wherein the conjugate is amphiphilically balancedsuch that it is aqueously soluble and able to penetrate biologicalmembranes.
 40. The mixture according to claim 1, wherein the oligomer iscovalently coupled to the drug.
 41. The mixture according to claim 1,wherein the oligomer further comprises a lipophilic moiety.
 42. Themixture according to claim 1, wherein the polyalkylene glycol moiety isa lower alkyl polyalkylene glycol moiety.
 43. The mixture according toclaim 42, wherein the lower alkyl polyalkylene glycol moiety is apolyethylene glycol moiety.
 44. The mixture according to claim 43,wherein the oligomer further comprises a lipophilic moiety.
 45. Themixture according to claim 42, wherein the lower alkyl polyalkyleneglycol moiety is a polypropylene glycol moiety.
 46. The mixtureaccording to claim 45, wherein the polypropylene glycol moiety isuniform.
 47. The mixture according to claim 46, wherein the oligomer isdevoid of a lipophilic moiety, and wherein the conjugate isamphiphilically balanced such that it is aqueously soluble and able topenetrate biological membranes.
 48. The mixture according to claim 1,wherein each conjugate comprises a plurality of oligomers.
 49. Themixture according to claim 48, wherein each oligomer in the plurality ofoligomers is the same.
 50. The mixture according to claim 1, wherein theoligomer comprises a first polyalkylene glycol moiety covalently coupledto the drug by a non-hydrolyzable bond and a second polyalkylene glycolmoiety covalently coupled to the first polyalkylene glycol moiety by ahydrolyzable bond.
 51. The mixture according to claim 1, wherein theoligomer further comprises a lipophilic moiety covalently coupled to thesecond polyethylene glycol moiety.
 52. The mixture according to claim 1,wherein the conjugates are each amphiphilically balanced such that eachconjugate is aqueously soluble and able to penetrate biologicalmembranes.
 53. A pharmaceutical composition comprising: the mixtureaccording to claim 1; and a pharmaceutically acceptable carrier.
 54. Amixture of conjugates each comprising a drug coupled to an oligomer thatcomprises a polyalkylene glycol moiety, said mixture having a molecularweight distribution with a standard deviation of less than about 22Daltons.
 55. The mixture according to claim 54, wherein the standarddeviation of the molecular weight distribution is less than about 14Daltons.
 56. The mixture according to claim 54, wherein the standarddeviation of the molecular weight distribution is less than about 11Daltons.
 57. The mixture according to claim 54, wherein the polyalkyleneglycol moiety is a lower alkyl polyalkylene glycol moiety.
 58. Themixture according to claim 57, wherein the lower alkyl polyalkyleneglycol moiety has at least 7 polyalkylene glycol subunits.
 59. Themixture according to claim 57, wherein the lower alkyl polyalkyleneglycol moiety is a polyethylene glycol moiety.
 60. The mixture accordingto claim 59, wherein the oligomer further comprises a lipophilic moiety.61. The mixture according to claim 57, wherein the lower alkylpolyalkylene glycol moiety is a polypropylene glycol moiety.
 62. Themixture according to claim 61, wherein the polypropylene glycol moietyis uniform.
 63. The mixture according to claim 62, wherein the oligomeris devoid of a lipophilic moiety, and wherein the conjugate isamphiphilically balanced such that it is aqueously soluble and able topenetrate biological membranes.
 64. The mixture according to claim 54,wherein the drug is a polypeptide selected from the group consisting ofadrenocorticotropic hormone peptides, adrenomedullin peptides,allatostatin peptides, amylin peptides, amyloid beta-protein fragmentpeptides, angiotensin peptides, antibiotic peptides, antigenicpolypeptides, anti-microbial peptides, apoptosis related peptides,atrial natriuretic peptides, bag cell peptides, bombesin peptides, boneGLA peptides, bradykinin peptides, brain natriuretic peptides,C-peptides, C-type natriuretic peptides, calcitonin peptides, calcitoningene related peptides, CART peptides, casomorphin peptides, chemotacticpeptides, cholecystokinin peptides, colony-stimulating factor peptides,corticortropin releasing factor peptides, cortistatin peptides, cytokinepeptides, dermorphin peptides, dynorphin peptides, endorphin peptides,endothelin peptides, ETa receptor antagonist peptides, ETh receptorantagonist peptides, enkephalin peptides, fibronectin peptides, galaninpeptides, gastrin peptides, glucagon peptides, Gn-RH associatedpeptides, growth factor peptides, growth hormone peptides, GTP-bindingprotein fragment peptides, guanylin peptides, inhibin peptides, insulinpeptides, interleukin peptides, laminin peptides, leptin peptides,leucokinin peptides, luteinizing hormone-releasing hormone peptides,mastoparan peptides, mast cell degranulating peptides, melanocytestimulating hormone peptides, morphiceptin peptides, motilin peptides,neuro-peptides, neuropeptide Y peptides, neurotropic factor peptides,orexin peptides, opioid peptides, oxytocin peptides, PACAP peptides,pancreastatin peptides, pancreatic polypeptides, parathyroid hormonepeptides, parathyroid hormone-related peptides, peptide T peptides,prolactin-releasing peptides, peptide YY peptides, renin substratepeptides, secretin peptides, somatostatin peptides, substance Ppeptides, tachykinin peptides, thyrotropin-releasing hormone peptides,toxin peptides, vasoactive intestinal peptides, vasopressin peptides,and virus related peptides.
 65. A mixture of conjugates each comprisinga drug coupled to a polymer comprising a polyalkylene glycol moiety,wherein the mixture has a dispersity coefficient (DC) greater than10,000 where${D\quad C} = \frac{\left( {\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}}} \right)^{2}}{{\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}^{2}{\sum\limits_{i = 1}^{n}\quad N_{i}}}} - \left( {\sum\limits_{i = 1}^{n}\quad {N_{i}M_{i}}} \right)^{2}}$

wherein: n is the number of different molecules in the sample; N_(i) isthe number of i^(th) molecules in the sample; and M_(i) is the mass ofthe i^(th) molecule.
 66. The mixture according to claim 65, wherein thedispersity coefficient is greater than 100,000.
 67. The mixtureaccording to claim 65, wherein the dispersity coefficient is greaterthan 500,000.
 68. The mixture according to claim 65, wherein thepolyalkylene glycol moiety is a lower alkyl polyalkylene glycol moiety.69. The mixture according to claim 68, wherein the lower alkylpolyalkylene glycol moiety has at least 7 polyalkylene glycol subunits.70. The mixture according to claim 68, wherein the lower alkylpolyalkylene glycol moiety is a polyethylene glycol moiety.
 71. Themixture according to claim 70, wherein the oligomer firther comprises alipophilic moiety.
 72. The mixture according to claim 68, wherein thelower alkyl polyalkylene glycol moiety is a polypropylene glycol moiety.73. The mixture according to claim 72, wherein the polypropylene glycolmoiety is uniform.
 74. The mixture according to claim 73, wherein theoligomer is devoid of a lipophilic moiety, and wherein the conjugate isamphiphilically balanced such that it is aqueously soluble and able topenetrate biological membranes.
 75. The mixture according to claim 65,wherein the drug is a polypeptide selected from the group consisting ofadrenocorticotropic hormone peptides, adrenomedullin peptides,allatostatin peptides, amylin peptides, amyloid beta-protein fragmentpeptides, angiotensin peptides, antibiotic peptides, antigenicpolypeptides, anti-microbial peptides, apoptosis related peptides,atrial natriuretic peptides, bag cell peptides, bombesin peptides, boneGLA peptides, bradykinin peptides, brain natriuretic peptides,C-peptides, C-type natriuretic peptides, calcitonin peptides, calcitoningene related peptides, CART peptides, casomorphin peptides, chemotacticpeptides, cholecystokinin peptides, colony-stimulating factor peptides,corticortropin releasing factor peptides, cortistatin peptides, cytokinepeptides, dermorphin peptides, dynorphin peptides, endorphin peptides,endothelin peptides, ETa receptor antagonist peptides, ETh receptorantagonist peptides, enkephalin peptides, fibronectin peptides, galaninpeptides, gastrin peptides, glucagon peptides, Gn-RH associatedpeptides, growth factor peptides, growth hormone peptides, GTP-bindingprotein fragment peptides, guanylin peptides, inhibin peptides, insulinpeptides, interleukin peptides, laminin peptides, leptin peptides,leucokinin peptides, luteinizing hormone-releasing hormone peptides,mastoparan peptides, mast cell degranulating peptides, melanocytestimulating hormone peptides, morphiceptin peptides, motilin peptides,neuro-peptides, neuropeptide Y peptides, neurotropic factor peptides,orexin peptides, opioid peptides, oxytocin peptides, PACAP peptides,pancreastatin peptides, pancreatic polypeptides, parathyroid hormonepeptides, parathyroid hormone-related peptides, peptide T peptides,prolactin-releasing peptides, peptide YY peptides, renin substratepeptides, secretin peptides, somatostatin peptides, substance Ppeptides, tachykinin peptides, thyrotropin-releasing hormone peptides,toxin peptides, vasoactive intestinal peptides, vasopressin peptides,and virus related peptides.
 76. A mixture of conjugates in which eachconjugate: comprises a drug coupled to an oligomer; and has the samenumber of polyalkylene glycol subunits.
 77. The mixture according toclaim 76, wherein the polyalkylene glycol moiety is a lower alkylpolyalkylene glycol moiety.
 78. The mixture according to claim 77,wherein the lower alkyl polyalkylene glycol moiety has at least 7polyalkylene glycol subunits.
 79. The mixture according to claim 77,wherein the lower alkyl polyalkylene glycol moiety is a polyethyleneglycol moiety.
 80. The mixture according to claim 79, wherein theoligomer further comprises a lipophilic moiety.
 81. The mixtureaccording to claim 77, wherein the lower alkyl polyalkylene glycolmoiety is a polypropylene glycol moiety.
 82. The mixture according toclaim 81, wherein the polypropylene glycol moiety is uniform.
 83. Themixture according to claim 82, wherein the oligomer is devoid of alipophilic moiety, and wherein the conjugate is amphiphilically balancedsuch that it is aqueously soluble and able to penetrate biologicalmembranes.
 84. The mixture according to claim 76, wherein the drug is apolypeptide selected from the group consisting of adrenocorticotropichormone peptides, adrenomedullin peptides, allatostatin peptides, amylinpeptides, amyloid beta-protein fragment peptides, angiotensin peptides,antibiotic peptides, antigenic polypeptides, anti-microbial peptides,apoptosis related peptides, atrial natriuretic peptides, bag cellpeptides, bombesin peptides, bone GLA peptides, bradykinin peptides,brain natriuretic peptides, C-peptides, C-type natriuretic peptides,calcitonin peptides, calcitonin gene related peptides, CART peptides,casomorphin peptides, chemotactic peptides, cholecystokinin peptides,colony-stimulating factor peptides, corticortropin releasing factorpeptides, cortistatin peptides, cytokine peptides, dermorphin peptides,dynorphin peptides, endorphin peptides, endothelin peptides, ETareceptor antagonist peptides, ETb receptor antagonist peptides,enkephalin peptides, fibronectin peptides, galanin peptides, gastrinpeptides, glucagon peptides, Gn-RH associated peptides, growth factorpeptides, growth hormone peptides, GTP-binding protein fragmentpeptides, guanylin peptides, inhibin peptides, insulin peptides,interleukin peptides, laminin peptides, leptin peptides, leucokininpeptides, luteinizing hormone-releasing hormone peptides, mastoparanpeptides, mast cell degranulating peptides, melanocyte stimulatinghormone peptides, morphiceptin peptides, motilin peptides,neuro-peptides, neuropeptide Y peptides, neurotropic factor peptides,orexin peptides, opioid peptides, oxytocin peptides, PACAP peptides,pancreastatin peptides, pancreatic polypeptides, parathyroid hormonepeptides, parathyroid hormone-related peptides, peptide T peptides,prolactin-releasing peptides, peptide YY peptides, renin substratepeptides, secretin peptides, somatostatin peptides, substance Ppeptides, tachykinin peptides, thyrotropin-releasing hormone peptides,toxin peptides, vasoactive intestinal peptides, vasopressin peptides,and virus related peptides.
 85. A mixture of conjugates in which eachconjugate has the same molecular weight and has the formula:

wherein: B is a bonding moiety; L is a linker moiety; G, G′ and G″ areindividually selected spacer moieties; R is a lipophilic moiety and R′is a polyalkylene glycol moiety, or R′ is the lipophilic moiety and R isthe polyalkylene glycol moiety; T is a terminating moiety; h, i, j, k, mand n are individually 0 or 1, with the proviso that when R is thepolyalkylene glycol moiety; m is 1, and when R′ is the polyalkyleneglycol moiety, n is 1; and p is an integer from 1 to the number ofnucleophilic residues on the drug.
 86. The mixture according to claim85, wherein the polyalkylene glycol moiety is a lower alkyl polyalkylenemoiety.
 87. The mixture according to claim 86, wherein the lower alkylpolyalkylene glycol moiety has at least 7 polyalkylene glycol subunits.88. The mixture according to claim 86, wherein the lower alkylpolyalkylene glycol moiety is a polyethylene glycol moiety.
 89. Themixture according to claim 88, wherein: R is the polyethylene glycolmoiety; R′ is a lipophilic moiety; n and m are 1; and i, j and k are 0.90. The mixture according to claim 88, wherein: R is a lipophilicmoiety; R′ is the polyethylene glycol moiety; n and m are 1; and i, jand k are each
 0. 91. The mixture according to claim 86, wherein thelower alkyl polyalkylene glycol moiety is a polypropylene glycol moiety.92. The mixture according to claim 91, wherein the polypropylene glycolmoiety is uniform.
 93. The mixture according to claim 92, wherein: R isthe polypropylene glycol moiety; m is 1; i, j, k and n are each 0; andeach conjugate in the mixture is amphiphilically balanced such that eachconjugate is aqueously soluble and able to penetrate biologicalmembranes.
 94. The mixture according to claim 85, wherein the drug is apolypeptide selected from the group consisting of adrenocorticotropichormone peptides, adrenomedullin peptides, allatostatin peptides, amylinpeptides, amyloid beta-protein fragment peptides, angiotensin peptides,antibiotic peptides, antigenic polypeptides, anti-microbial peptides,apoptosis related peptides, atrial natriuretic peptides, bag cellpeptides, bombesin peptides, bone GLA peptides, bradykinin peptides,brain natriuretic peptides, C-peptides, C-type natriuretic peptides,calcitonin peptides, calcitonin gene related peptides, CART peptides,casomorphin peptides, chemotactic peptides, cholecystokinin peptides,colony-stimulating factor peptides, corticortropin releasing factorpeptides, cortistatin peptides, cytokine peptides, dermorphin peptides,dynorphin peptides, endorphin peptides, endothelin peptides, ETareceptor antagonist peptides, ETb receptor antagonist peptides,enkephalin peptides, fibronectin peptides, galanin peptides, gastrinpeptides, glucagon peptides, Gn-RH associated peptides, growth factorpeptides, growth hormone peptides, GTP-binding protein fragmentpeptides, guanylin peptides, inhibin peptides, insulin peptides,interleukin peptides, laminin peptides, leptin peptides, leucokininpeptides, luteinizing hormone-releasing hormone peptides, mastoparanpeptides, mast cell degranulating peptides, melanocyte stimulatinghormone peptides, morphiceptin peptides, motilin peptides,neuro-peptides, neuropeptide Y peptides, neurotropic factor peptides,orexin peptides, opioid peptides, oxytocin peptides, PACAP peptides,pancreastatin peptides, pancreatic polypeptides, parathyroid hormonepeptides, parathyroid hormone-related peptides, peptide T peptides,prolactin-releasing peptides, peptide YY peptides, renin substratepeptides, secretin peptides, somatostatin peptides, substance Ppeptides, tachykinin peptides, thyrotropin-releasing hormone peptides,toxin peptides, vasoactive intestinal peptides, vasopressin peptides,and virus related peptides.
 95. A process for synthesizing asubstantially monodispersed mixture of conjugates each conjugatecomprising a drug coupled to an oligomer that comprises a polyethyleneglycol moiety, said process comprising: reacting a substantiallymonodispersed mixture comprising compounds having the structure ofFormula I: R¹(OC₂H₄)_(m)—O⁻X⁺  (I) wherein R¹ is H or a lipophilicmoiety; m is from 1 to 25; and X⁺ is a positive ion, with asubstantially monodispersed mixture comprising compounds having thestructure of Formula II: R²(OC₂H₄)_(n)—OMs  (II) wherein R² is H or alipophilic moiety; and n is from 1 to 25, under conditions sufficient toprovide a substantially monodispersed mixture comprising polymers havingthe structure of Formula III: R²(OC₂H₄)_(m+n)—OR¹  (III); activating thesubstantially monodispersed mixture comprising polymers of Formula IIIto provide a substantially monodispersed mixture of activated polymerscapable of reacting with a drug; and reacting the substantiallymonodispersed mixture of activated polymers with a substantiallymonodispersed mixture of drugs under conditions sufficient to provide asubstantially monodispersed mixture of conjugates each comprising a drugcoupled to an oligomer that comprises a polyethylene glycol moiety withm+n subunits.
 96. The process according to claim 95, wherein R² is afatty acid moiety or an ester of a fatty acid moiety.
 97. The processaccording to claim 96, wherein the fatty acid moiety or the ester of afatty acid moiety comprises an alkyl moiety at least 5 carbon atoms inlength.
 98. The process according to claim 95, wherein R¹ is a methylgroup.
 99. The process according to claim 95, further comprising:reacting a substantially monodispersed mixture comprising compoundshaving the structure of Formula V: R²(OC₂H₄)_(n)—OH  (V) with amethanesulfonyl halide under conditions sufficient to provide asubstantially monodispersed mixture comprising compounds having thestructure of Formula II: R²(OC₂H₄)_(n)—OMs  (II).
 100. The processaccording to claim 95, further comprising: reacting a substantiallymonodispersed mixture comprising compounds having the structure ofFormula VI: R²—OMs  (VI) wherein R² is a lipophilic moiety; with asubstantially monodispersed mixture comprising compounds having thestructure of Formula VII: R³(OC₂H₄)_(m)—O⁻X₂ ⁺  (VII) wherein R³ isbenzyl, trityl, or THP; and X₂ ⁺ is a positive ion; under conditionssufficient to provide a substantially monodispersed mixture comprisingcompounds having the structure of Formula VIII:R³(OC₂H₄)_(m)—OR²  (VIII); and reacting the substantially monodispersedmixture comprising compounds having the structure of Formula VIII underconditions sufficient to provide a substantially monodispersed mixturecomprising compounds having the structure of Formula V:R²(OC₂H₄)_(m)—OH  (V).
 101. The process according to claim 95, furthercomprising: reacting a substantially monodispersed mixture comprisingcompounds having the structure of Formula IV: R¹(OC₂H₄)_(n)—OH  (IV)under conditions sufficient to provide a substantially monodispersedmixture comprising compounds having the structure of Formula I:R¹(OC₂H₄)_(n)—O⁻X⁺  (I).
 102. The process according to claim 95, whereinthe activating of the substantially monodispersed mixture comprisesreacting the substantially monodispersed mixture of polymers of FormulaIII with N-hydroxy succinimide to provide an activated polymer capableof reacting with a drug.
 103. The process according to claim 95, whereinthe drug is a polypeptide, and wherein the reacting of the substantiallymonodispersed mixture of activated polymers with a substantiallymonodispersed mixture of polypeptides comprises: reacting thesubstantially monodispersed mixture of activated polymers with one ormore amino functionalities of the polypeptide to provide a substantiallymonodispersed mixture of conjugates each comprising the polypeptidecoupled to an oligomer that comprises a polyethylene glycol moiety withm+n subunits.